Cell, Vol. 61, 203-212,

April 20, 1990, Copyright

0 1990 by Cell Press

Signal Transduction by Receptors with Tyrosine Kinase Activity Axe1 Ullrich’ and Joseph Schlessingert Max-Planck-lnstitut fur Biochemie Am Klopferspitz 18A 8033 Martinsried Federal Republic of Germany t Department of Pharmacology New York University Medical Center New York, New York 10016 ??

Polypeptides such as growth factors, differentiation factors, and hormones are crucial components of the regulatory system that coordinates development of multicellular organisms. Many of these factors mediate their pleiotropic actions by binding to and activating cell surface receptors with an intrinsic protein tyrosine kinase activity. Growth factor receptors with protein tyrosine kinase activity, or receptor tyrosine kinases, have a similar molecular topology. All possess a large glycosylated, extracellular ligand binding domain, a single hydrophobic transmembrane region, and a cytoplasmic domain that contains a tyrosine kinase catalytic domain (Hanks et al., 1988; Yarden and Ullrich, 1988; Schlessinger, 1988; Williams, 1989). Because of their configuration, receptor tyrosine kinases can be envisioned as membrane-associated allosteric enzymes. Unlike water-soluble allosteric enzymes, receptor tyrosine kinases have a topology dictating that the ligand binding domain and protein tyrosine kinase activity are separated by the plasma membrane. Therefore, receptor activation due to extracellular ligand binding must be translated across the membrane barrier into activation of intracellular domain functions. On the basis of sequence similarity and distinct structural characteristics, it is possible to classify these receptors into subclasses (Figure 1). Characteristic structural features of the subclasses include two cysteine-rich repeat sequences in the extracellular domain of monomeric subclass I receptors, disulfide-linked heterotetrameric a& structures with similar cysteine-rich sequences in subclass II receptors, and five or three immunoglobulin-like repeats in the extracellular domains of subclass III and IV receptors, respectively. The tyrosine kinase domain of the latter is interrupted by hydrophilic insertion sequences of varying length. The availability of receptor tyrosine kinase cDNA clones has made it possible to initiate detailed structure-function analyses of the mechanisms of action of receptor tyrosine kinase family members. Numerous mutants of the insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-l), colony-stimulating factor 1 (CSF-l), and other receptors have been generated and studied. In this review we describe recent studies that utilize this powerful approach to address major mechanistic questions of receptor action and signal transduction, with particular emphasis on the EGF receptor.

Review

Receptor Dimerization and Activation It appears that ligand-induced activation of the kinase domain and its signaling potential are mediated by receptor oligomerization (Schlessinger, 1988, and references therein; but see also Williams, 1989). Ligand binding and the subsequent conformational alteration of the extracellular domain induce receptor oligomerization, which stabilizes interactions between adjacent cytoplasmic domains and leads to activation of kinase function by molecular interaction. Receptor oligomerization permits the transmission of a conformational change from the extracellular domain to the cytoplasmic domain without requiring alterations in the positioning of amino acid residues within the transmembrane domain. Receptor oligomerization is a universal phenomenon among growth factor receptors. It has been detected in living cells, isolated membranes, and preparations of solubilized and purified receptors (Yarden and Schlessinger, 1987a, 198713;Cachet et al., 1988; and references therein). It may be induced by monomeric ligands, such as EGF, that induce conformational changes (Greenfield et al., 1989) resulting in receptor-receptor interactions, or bivalent ligands, such as PDGF and CSF-1, that mediate dimerization of neighboring receptors (Seifert et al., 1989; Heldin et al., 1989; Hammacher et al., 1989). Oligomerized growth factor receptors possess elevated protein tyrosine kinase activity and enhanced ligand binding affinity (Yarden and Schlessinger, 1987a, 1987b; Boni-Schnetzler and Pilch, 1987). The heterotetrameric structure of subclass II receptors for insulin and IGF-1 represents a variation on this theme. Here, ligand binding induces allosteric interaction of two a8 halves within the disulfide bridge-stabilized receptor complex (Figure 2). The universality of this dimer-mediated receptor activation mechanism for all receptor tyrosine kinase subclasses and the conservation of the structural parameters involved has been demonstrated by the construction of fully functional chimeric receptors consisting of major domains of different receptor subclasses (Riedel et al., 1989, and references therein). The dimerization mechanism further implies the possible existence of hybrid complexes between structurally very similar receptors such as a and 8 type PDGF receptors, EGF receptor and HER2/neu, or insulin and IGF-1 receptors. In some cases heterodimer formation has already been demonstrated (Hammacher et al., 1989; Soos and Siddle, 1989) (Figure 2). Direct proof that such hybrid receptors are functional and may even generate distinct signals is not yet available. For subclass III receptors, which bind dimerit ligands, further possibilities for the generation of modulated signals are provided by interaction with heterodimeric ligands such as PDGF-AB. Receptor tyrosine kinases catalyze the phosphorylation of exogenous substrates as well as tyrosine residues within their own polypeptide chains. In the case of the

Cell 204

Subclass I ?? + Ligand

*

Subclass II

s s

a +

Ligand

*

1

PTK

Subclass III EGF-R HERZlneu HER3/c-erbB3 Xmrk

Insulin-R IGF-I-R IRR

Figure 1. Schematic Representation classes

PDGF-R-A PDGF-R-0 -F&R

FGF-R f/g bek

0 + Ligand Dimer +

of Receptor Tyrosine Kinase Sub-

The previously established classification (Yarden and Ullrich, 1988) has been extended by recently described receptor tyrosine kinases such as HER3/c-erbB-3 (Kraus et al., 1989) IRR (Shier and Watt, 1989) Xmrk (Wittbrodt et al., 1989) and the receptors for acidic FGF and basic FGF, termed g and bek (Ruta et al., 1989; Pasquale and Singer, 1989; and references therein). KI = kmase insert region.

insulin receptor, ligand-induced autophosphorylation of several tyrosine residues within the 8 subunit cytoplasmic domain increases the V,,, of the kinase activity, which maintains the protein tyrosine kinase in the activated state, even in the absence of bound ligand (Rosen et al., 1983). Autophosphorylation of the PDGF receptor leads to the phosphorylation of a consensus tyrosine residue (Tyr857) present in the catalytic domain of all tyrosine kinases, as well as Tyr751, which lies in the kinase insert and appears to be involved in modulating the interaction between activated receptors and cellular proteins (Kazlauskas and Cooper, 1989). EGF receptor autophosphorylation sites are clustered at the carboxy-terminal tail (Yarden and UIIrich, 1988, and references therein; Margolis et al., 1989a) and also appear to modulate the interaction of activated EGF receptors with substrates and other proteins (Bertics et al., 1988; Honegger et al., 1988a, 1988b; Margolis et al., 1990a). The autophosphorylation sites of the EGF receptor compete with exogenous substrates for the substrate binding site of the tyrosine kinase domain. Hence, autophosphorylation appears to release an internal constraint by establishing a conformation of the receptor that is competent to interact with and phosphorylate cellular substrates (Honegger et al., 1988a, 1988b). That autophosphorylation of EGF and insulin receptors can occur by intermolecular cross-phosphorylation in vitro and in living cells (Honegger et al., 1989, 1990a; Ballotti et al., 1989) further supports the importance of. receptor oligomerization in the process of receptor activation. Ligand Binding While the extracellular domains of the insulin receptor (a

=A!& 4% =s!&

Figure 2. Models of Receptor Subclass-Specific Mechanism of Activation by Dimerization

Variations

of the

Receptor activation may occur by binding of monomeric ligands resulting in a conformational change of the extracellular domain and dimer formation (subclass I), by interaction of the ligand with a disulfidestabilized receptor dimer and subsequent intracomplex conformational change (subclass II), or by mediation of dimer formation through a dimeric ligand (subclass Ill).

subunit) and EGF receptor share homologous cysteinerich sequence repeat domains (Figure l), the extracellular ligand binding domains of the PDGF family of receptors (subclass Ill) have an immunoglobulin-like structure and apparently have evolved convergently from distinct ancestral genes. Similar to the five repeats of homologous sequences in the PDGF receptor extracellular domain (Yarden and Ullrich, 1988, and references therein), the members of the fibroblast growth factor (FGF) receptor family (subclass IV), bek and f/g (Ruta et al., 1989; Pasquale and Singer, 1989; and references therein), exhibit three related sequence repeats in their extracellular domains. These repeats show weak but significant homology with the corresponding region of the interleukin 1 receptor (Ruta et al., 1989) a member of the immunoglobulin superfamily of cell surface polypeptides. While the functional significance of the cysteine-rich and immunoglobulin-like structures is unknown, progress has been made toward the characterization of the ligand binding region of EGF and insulin receptors. Affinity labeling experiments with lz51-EGF indicated that a region flanked by the two cysteine-rich domains of the EGF receptor (denoted domain III; Figure 3) includes major structural determinants of the EGF binding site. These results were supported by a functional analysis of the ligand binding domain using chimeric human-chicken receptors (Lax et al., 1989). Since the chicken EGF receptor is highly homologous to its human counterpart and binds EGF with w250-fold lower affinity, it was possible to

Review: Receptor Tyrosine 205

Kinases

DOMAIN

FUNCTION

Extracellular

Ligand binding Dimerization

Transmembrane

Membrane anchor

Frgure 3. Proposed Structure-Function ogy of the EGF Receptor

Juxtamembrane

Negative control (Thr 654)

Tyrosine Kinase

ATP binding (Lys 721) Substrate binding Catalytic activities

Carboxy-terminal tail

Signal regulation

generate

chimeric

receptor

molecules

tion of the human

EGF receptor

the higher

binding

chicken

affinity

EGF receptor

EGF receptor

characteristics.

containing

EGF receptor.

molecule

EGF receptor the human

domain

Moreover,

containing

bound

references

structures therein),

organization

(Figure

and close

this model, formed

tween

binding

Attempts model.

responds

is common

to

in many allosteric

thus allowing

be-

transfer

involved

of an

in formwith

of photoaffinity-labeled

the amino-terminal receptor

I of the EGF receptor,

this

tryptic

region (amino

a subunit,

et al., 1989). This suggests of receptor

(Weiner et al., 1989). Yet, it appears

signal transduction. consisting

ceptors

which cor-

in ligand

bind-

that, analogous

this region contributes

binding

of combinations protein,

Region

The neu proto-oncogene gle valine stituted

residue

1988, and references activation

on their signaling ligand across

membrane

were able to interact

irrespective

sequence;

plasmic

domain.

regions of two mutant

receptors

receptors,

hydrophobic

by charged

residues

mains

could

amino

(Kashles

abolished

impair

acids

ligand-induced

the possibility

activation

structure

of a conformational

neu may have a stabilizing resulting

in dimerization signaling.

membrane

region plays

but do not exclude change

in this region.

effect

mutation

and thus constitutive

activation

environment

thereby

with internal

of

of the trans-

would be to anchor the receptor membrane,

in

on this conformation,

Thus, the main function

domain

of

of do-

and responsive-

to our model, the transmembrane

receptor

of the

with those

this discrepancy.

role in signal transduction,

According

sub-

inexact swapping

We favor the idea that the transmembrane a passive

were

that replacement

However,

receptor

explain

or

et al., 1988). In con-

region of the PDGF receptor

kinase activity.

bear-

The transmembrane

were either shortened

(1989) reported

of other receptors tyrosine

various

amino acids; in two other

mutant

Williams

that EGF was

and stimulate

regions.

stituted trast,

do-

by the cyto-

mutant EGF receptors

by three hydrophobic

transmembrane

of the transmem-

it was shown

the kinase activity

ing altered transmembrane

and

the plasma

of the transmembrane

in cells expressing

extended

nature

binding

on the signal generated

Moreover,

able to enhance responses

of the

the identity

main had no influence

capacity

et al., 1989; J. Lee et al.,

in the

connecting compartments

the of

the cell. product

is activated

in its transmembrane

by a glutamic

and IGF-1 re-

of the transmem-

1989). In all cases, the heterologous

extracellular Transmembrane

that the origin

kinase brane

intracellu-

of the EGF receptor,

and the PDGF, insulin,

et al., 1989; Lammers

for

receptors

of the extracellular,

brane region had no influence

domains

that the in-

is not essential

with chimeric

domains

demonstrated

plane of the plasma

characteristics.

region

Experiments

lar, and transmembrane

ness and might

are consistent

III of the EGF receptor,

to the definition

According

alters the interaction

pocket

of the insulin

to domain

to domain

growth

might lie in the cleft

the structures

binding

has implicated

ing (Wedekind

to interact

transition.

to characterize

acids 20-120)

most, if not

that are formed

membrane.

region

subunits,

Partial sequencing

peptides

to contribute

Ill, to-

or transforming

binding

conformational

ing the insulin

domain

Ill and I. Such a configuration

region

ligand

neighboring

allosteric

for the

regions would be in contact with each

domains

where

do-

of the EGF recep-

the two domains

the EGF binding

for a ligand

micro-

model

3). In this model,

to the plasma

between

enzymes

portion

in

1988, and

extracellular

a four-domain

its ligand-EGF

by the cysteine-rich

as

Subdomains II and IV (stippled) represent the cysteine-rich regrons of the extracellular domain. Most of the structural determinants that define EGF binding affinity are proposed to be located in the cleft formed by subdomains I and III. Left: side view. Right: top view. The symbols Sand R within the tyrosine kinase domain represent proposed interaction sites for substrates and regulatory factors.

of the transmembrane

HER2/neu

homology

Ullrich,

that enable the receptor

factor a (TGFa)-while other

and

merization tegrity

(Riedel

the internal

I, is proposed

all, of the determinants with

than

I and Ill of human

of the purified

of the extracellular

gether with domain

affinity

and initial data from electron

main of the EGF receptor,

specifically

Ill of the human

(Lax et al., 1989). (Yarden

characterization

tor is proposed

a

a chicken-human

domains

On the basis of these studies,

scopic

for

EGF with nearly the same affinity

EGF receptor

the primary

the por-

Specifically,

had only a 3-fold lower binding

the human chimeric

to identify

that was responsible

Topol-

acid residue therein).

of the protein tyrosine

al., 1988) which is essential

when a sin-

region

(Yarden

This mutation

Juxtamembrane The transmembrane

leads to the

is separated

kinase function

for the transforming

of neu and is likely to be due to enhanced

is sub-

and Ullrich, (Stern et

tamembrane

potential

ceptor

receptor

oligo-

Domain domain

of receptor

from the cytoplasmic, sequences

subclasses

the same subclass

tyrosine

catalytic

that are divergent

but conserved (Figure

between

kinases

domain by juxbetween

re-

members

of

3). A great deal of evidence

Cell 206

suggests that this domain is involved in modulation of receptor functions by heterologous stimuli, a process termed receptor transmodulation. Activation of PDGF or bombesin receptors by their respective ligands leads to the abolishment of high affinity EGF binding sites in a single cell. The same effect is achieved when protein kinase C (PKC) is activated, triggering phosphorylation of the EGF receptor on serine and threonine residues. One of the PKC target residues is Thr654, which is located in the juxtamembrane domain (Figure 3; Yarden and Ullrich, 1988, and references therein). Since both bombesin and PDGF are known to stimulate the phosphatidylinositol signaling pathway, it was proposed that their effect on EGF binding affinity is mediated by PKC. Hence, PKC may act as a mediator of receptor transmodulation and thus provide negative feedback for receptor activity control (Lin et al., 1986). While PKC-induced phosphorylation of EGF receptor results in inhibition of its kinase activity (Davis, 1988), similar to the insulin receptor (see Czech, 1989, and references therein), it appears to be essential also for the abolition of high affinity EGF binding (Livneh et al., 1987). Phosphorylation of Thr654 may not play a crucial role in the modulation of receptor affinity (Livneh et al., 1988; Davis, 1988; see also Lin et al., 1986); nevertheless, phosphorylation at this site seems to be involved in the control of mitogenic signaling by the receptor (Livneh et al., 1988). Interestingly, the phorbol ester PMA is also able to modulate the binding affinity of EGF in a chimeric receptor molecule composed of the extracellular domain of the EGF receptor fused to the transmembrane and cytoplasmic domains of HER2/neu, suggesting that the binding affinity of HER2/neu for its as yet unidentified ligand is also controlled by PKC-mediated transmodulation (J. Lee et al., 1989). The importance of the juxtamembrane region is further emphasized by a mutant insulin receptor containing phenylalanine in place of Tyr960. This alteration abolishes this receptor’s ability to phosphorylate the cellular substrate ~185 without affecting the overall level of receptor autophosphorylation (White et al., 1988). Protein Tyrosine Kinase Domain The tyrosine kinase domain is the most highly conserved portion of all receptor tyrosine kinase molecules (Figure 3). Among other highly conserved sequences of unknown function, the tyrosine kinase domain contains a consensus sequence, GlyXGlyXXGlyX(15-20)Lys (Yarden and Ullrich, 1988; Schlessinger, 1988; Hanks et al., 1988), that functions as part of the binding site for ATP. Replacement of the consensus lysine residue of the ATP binding site in the EGF, insulin, and PDGF receptors completely abolished their kinase activities both in vitro and in living cells (Chou et al., 1987; McClain et al., 1987; Russell et al., 1987; Honegger et al., 1987a; Chen et al., 1987; Williams, 1989). Additional receptor mutants generated to explore the role of protein tyrosine kinase activity include linker insertion mutants at different positions of the catalytic region (Prywes et al., 1986; Livneh et al., 1987);deletion mutants (Livneh et al., 1986), and a double-mutant EGF receptor in which both Lys721 and Thr654 were altered (Chen et al.,

1987; Glenneyet al., 1988). While the kinase activity of the various receptors was dispensable for their expression and targeting to the cell surface, it was indispensable for signal transduction and induction of both early and delayed cellular responses, including mitogenesis and transformation. Although normal in its binding characteristics, the kinase-negative mutant of the EGF receptor was unable to stimulate calcium influx, inositol phosphate formation, Na+/H+ exchange, c-fos and c-myc expression, S6 ribosomal protein phosphorylation, DNA synthesis, and transformation (Honegger et al., 1987a, 1987b; Chen et al., 1987; Moolenaar et al., 1988). This suggests that all receptor tyrosine kinase signaling activities depend on a functional tyrosine kinase and that these processes are mediated by tyrosine phosphorylation of cellular substrates. Surprisingly, the protein tyrosine kinase activity of the EGF receptor was also found to be essential for its targeting to lysosomes upon ligand-induced activation (Honegger et al., 1987a; Glenney et al., 1988; Chen et al., 1989; Felder et al., 1990). Whether this means that lysosomal targeting requires phosphorylation of a yet unidentified substrate(s) is not known. Alternatively, components of the intracellular routing system may only recognize the conformation of an activated receptor (Honegger et al., 1987a; Felder et al., 1990). Because of the multiple activities in which the tyrosine kinase domain is involved, one may postulate the existence of several distinct sites of interaction with substrates, regulatory factors, and cellular components involved in protein transport (Figure 3). Kinase Insertion Sequences The kinase domain of subclass III and IV receptor tyrosine kinases is divided into two halves by insertions of up to 100 mostly hydrophilic amino acid residues (Figure 1). The kinase inserts of the various receptors vary in length and show only marginal similarity. For a specific receptor, however, kinase insertion sequences are highly conserved between species, which suggests that they play an important role in receptor function. Escobedo and Williams (1988) reported that a deletion mutant lacking 82 amino acids of the PDGF receptor kinase insert lost its mitogenic signaling potential for CHO cells but was still able to stimulate various early responses. However, contrary results were obtained with a nearly identical PDGF receptor deletion mutant (A83) in another cell system, as well as with an analogous mutant of the CSF-1 receptor (Taylor et al., 1989). These latter studies demonstrated that most of the kinase insert region is dispensable for kinase activity and mitogenic signaling. The PDGF receptor kinase insert contains an autophosphorylation site (Tyr751). Mutational analysis suggested that autophosphorylation at this site regulates interactions with cellular substrates (Kazlauskas and Cooper, 1989). According to this view, autophosphorylation of Tyr751 in the kinase insert triggers the binding of the activated PDGF receptor to cellular proteins such as phosphatidylinositol3-kinase, whose activity is modulated by PDGF. It thus appears that the role of the kinase insert region is to modulate receptor interactions with certain cellular substrates and effector proteins.

Review: Receptor Tyrosine Kinases 207

tiated oncogenic capacity and increased the host range but did not provide a major oncogenic lesion (Khazaie et al., 1988). Similar experiments in other cell systems (Haley et al., 1989; Velu et al., 1989) and with other receptors, such as that for CSF-1, support the notion that carboxyterminal tail sequences exert negative control on receptor tyrosine kinase signaling functions (Roussel et al., 1987).

EGF

CA+2

Frgure 4. Potential Mechamsm Underlying Activahon of the Phosphatrdylmositol Signakng Pathway by Receptor Tyrosine Kinases Receptors wrth tyrosine kinase activity phosphorylate phospholrpase C-y (PLCy), possibly without the involvement of G proteins (Gp). Tyrosine phosphorylation of phospholipase C-y will activate the enzyme, thus linking tyrosine kinase activity and the phosphoinosrtol effector system. PMA = phorbol 12-myristate 13-acetate; PIP? = phosphatidylrnositol 4,Sbisphosphate; DG = diacylglycerol; IP, = inosrtol 1,4,5Wsphosphate.

The Carboxy-Terminal Tail The carboxy-terminal tail sequences are among the most divergent between all known receptor tyrosine kinases (Yarden and Ullrich, 1988). Several autophosphorylation sites have been mapped in this region of the EGF receptor, HER2/neu protein (Margolis et al., 1989a; Hazan et al., 1990) and insulin receptor (Tornqvist and Avruch, 1988). These tyrosine residues are conserved in the carboxyterminal tails within each receptor tyrosine kinase subclass (Yarden and Ullrich, 1988). The biological role of the carboxy-terminal tail and its autophosphorylation sites was explored using a series of EGF receptor mutants in which individual tyrosine autophosphorylation sites were replaced by phenylalanine residues (Bertics et al., 1988; Honegger et al., 1988a, 1988b; Khazaie et al., 1988). These mutant receptors had enzymatic and biological properties similar to wild-type EGF receptors expressed in the same cellular background (Honegger et al., 1988a, 1988b). EGF receptor autophosphorylation does not affect the Vmaxof the receptor kinase, and it slightly decreases the apparent K, for peptide substrates. These observations are consistent with a mechanism in which exogenous substrates and intrinsic autophosphorylation sites compete for the protein tyrosine kinase active site (Honegger et al., 1988a, 1988b). Autophosphorylation (or deletion of intrinsic sites) would remove the competitive potential of endogenous sequences, providing cellular substrates with increased access to binding sites. The carboxy-terminal tail of the EGF receptor may possess enough length and flexibility to interact with the substrate binding sites of the protein tyrosine kinase region and thus modulate its capacity to interact with exogenous substrates. Consistent with this model, cells expressing mutant EGF receptors with altered autophosphorylation sites were mitogenically responsive to lower doses of EGF than cells expressing similar levels of wild-type receptors (Honegger et al., 1988b). Deletions in the carboxy-terminal tail had a similar effect on transformation: they poten-

Multiple Pathways of Signal Transduction Growth factors trigger an array of cellular responses. These include stimulation of Na+/H+ exchange, Ca2+ influx, activation of phospholipase C-T, and stimulation of glucose and amino acid transport. The stimulation of phospholipase C-T leads to the generation of phosphatidylinositol metabolites, such as inositol 1,4,5-trisphosphate, which cause the release of Ca*+ from intracellular compartments and the generation of diacyglycerol, the natural activator of PKC (Nishizuka, 1988; Figure 4). Numerous intrinsic cellular substrates require phosphotyrosine by the action of receptor tyrosine kinases. Addition of growth factor also stimulates the phosphorylation of multiple substrates on serine and threonine residues. Some of these latter substrates are phosphorylated by PKC (Figure 4) by S6 kinase, or by other, as yet unidentified serinel threonine kinases. It is assumed that the phosphorylation of cellular substrates, together with alterations in the ionic content of the cell, provides an internal stimulus for cell growth. However, the chain of events that is initiated by tyrosine phosphorylation of cellular substrates is still poorly understood Several receptor tyrosine kinase substrates of potential biological importance have been identified (Figure 5). Both PDGF and EGF can induce tyrosine phosphorylation of phospholipase C-y in vitro and in living cells (Margolis et al., 1989b; Meisenhelder et al., 1989; Wahl et al., 1989). In addition, phospholipase C-y was observed to associate with the activated receptor kinases in a ligand- and kinase-dependent manner (Margolis et al., 1989b. 1990; Kumjian et al., 1989). While tyrosine phosphorylation has not yet been shown to activate phospholipase C-y activity directly, much evidence suggests that this is indeed the case (Moolenaar et al., 1988; Margolis et al., 1989b; Meisenhelder et al., 1989). Overexpression of phospholipase C-y in transfected cells leads to a dramatic enhancement of phospholipase C activity and overproduction of inositol 1,45-trisphosphate. Surprisingly, the PDGF-induced Ca*+ signal (from internal stores) and DNA synthesis were not increased in the cells overexpressing phospholipase C-y (Margolis et al., 1990a, 1990b). In addition, despite the similarities between the PDGF and CSF-1 receptors (Figure l), the binding of CSF-1 to its cognate receptor leads to stimulation of DNA synthesis without phospholipase C-y phosphorylation and enhanced phosphatidylinositol metabolism (Downing et al., 1989). Similarly, insulin and IGF-1 receptors can mediate a mitogenic response in NIH 3T3 cells equal to that observed with serum, without phosphorylation of PLC-y (Lammers et al., 1989; Nishibe et al., 1990). Taken together, these data suggest that PDGF-induced inositol 1,4,5-trisphosphate generation is not the sole

Cell 208

Frgure 5. Receptor-Mediated

Multiple Signaling

Pathways

Direct phosphorylation (black dots on symbols) of substrates phospholipase C-y (PLQ). phosphatidylinositol 3-kinase (Ptdlns-SK). ras GTPase-activating protein (GAP), and raf leads to secondary events, including enzymatic activation and metabolite formation, activation of enzymatic functions by association, and serinenhreonine phosphorylation (white dot on symbol) of substrates. DAG = diacylglycerol; IPs = inositol 1,4,5-trisphosphate; Ptdlns(3)P = phosphatidylinositol 3-phosphate.

mechanism underlying PDGF-induced DNA synthesis. These results are also compatible with the notion that the phosphatidylinositol signaling pathway does not play an essential role in the mitogenic response (Lopez-Rivas et al., 1987; Wlemain et al., 1989; Margolis et al., 1990b). Reports from several laboratories provide good evidence that phosphatidylinositol 3-kinase (Varticovski et al., 1989) and the ras GTPase-activating protein (Molloy et al., 1989) are direct substrates for some receptor tyrosine kinases (Figure 5). It has also been suggested that the c-r& proto-oncogene product becomes phosphorylated in response to PDGF receptor activation (Morrison et al., 1989). It is intriguing that the proteins identified thus far as receptor tyrosine kinase targets are all either components of second messenger pathways, proto-oncogene products, or factors that regulate the activity of protooncogene products. Several experimental approaches indicate that these receptor tyrosine kinase target molecules are components of a preexisting complex or assembly of molecules involved in activation of diverse and distinct signaling pathways that lead to pleiotropic cellular responses (Margolis et al., 1989b) (Figures 5 and 6). Such a’signal transfer particle” (Figure 6) may exist even in the absence of ligand, when the receptor is inactive. Receptor activation may promote the interaction with other factors and thereby change the composition of the signal transfer particle. This has been demonstrated for phospholipase C-y, whose association with either PDGF or EGF receptor is dependent on an activated kinase (Figure 6; Margolis et al., 1989h 1990a). Cellular Sorting of Receptor Tyrosine Kinases Following ligand binding, receptor aggregates present in coated pits are rapidly internalized. Subsequently, receptors such as those for EGF and insulin enter distinct cellular sorting pathways leading to either degradation or recycling to the cell surface, respectively. The structural determinants that define the distinct targeting of these

STPi Figure 6. Schematic

STPa Structure of Proposed Signal Transfer Particles

Regulatory factors, which could also represent substrates (dark shading), are bound to the inactive tyrosine kinase domain of the receptor, forming the inactive signal transfer particle (STPi). Activation by ligand stimulation may lead to dissociation of regulatory factors that may be phosphorylated on tyrosine residues (black dots), and the newly formed active conformation presents a binding site(s) for substrates such as phospholipase C-T (PLCT), ras GTPase-activating protein (GAP), phosphotidylinositol 3-kinase (Pl3K). and the raf proto-oncogene product. Some of these substrates may be preformed as a substrate complex or could individually compete for the substrate bindrng site. Substrate association with the activated receptor results in the active signal transfer particle (STPa), which triggers a cascade of molecular events leading to the activation of a pleiotroprc signal required for the cellular resoonse.

receptors are contained in their cytoplasmic domains, as shown in experiments employing chimeric EGF-insulin receptor molecules (Riedel et al., 1989). The kinase activity of these receptors is indispensable for their targeting to lysosomes. Kinase-deficient EGF and insulin receptor mutants fail to undergo typical ligandinduced down-regulation (McClain et al., 1987; Russell et al., 1987; Honegger et al., 1987a, 1990b; Glenney et al., 1988; Chen et al., 1989). The kinase-negative EGF receptor mutant is internalized at nearly the same rate as the normal receptor but is rapidly recycled to the cell surface for reutilization (Honegger et al., 1987a, 1990b). Even when coexpressed in the same cell as wild-type EGF receptors, EGF stimulates the down-regulation and degradation of only the wild-type receptor, while the kinasenegative mutant recycles back to the cell surface for reutilization (Honegger et al., 1990a). Hence, intracellular trafficking of EGF receptors must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity. Moreover, kinase-positive EGF receptor mutants devoid of all four autophosphorylation sites are degraded in the same manner as normal EGF receptors, indicating that autophosphorylation is not the signal for receptor down-regulation and degradation. Felder et al. (1990) recently compared the pathways of internalization of wild-type and kinase-negative EGF receptor mutants by utilizing electron microscopy and finestructure analysis by immunocytochemistry. The pathways of internalization of wild-type and kinase-negative mutants were found to be indistinguishable for the first lo-20 min, until the receptors reached multivesicular bod-

Review: Receptor Tyrosine 209

Kinases

ies. At the multivesicular body, wild-type receptors were localized in small vesicles in the lumen. However, the kinase-negative mutant receptors, destined for recycling to the plasma membrane, were localized to the limiting membrane and surface protrusions of the multivesicular body. It appears, therefore, that kinase activity provides a sorting signal, perhaps by tyrosine phosphorylation of “sorting substrates;’ which leads to spatial segregation of wild-type receptor from a constitutive recycling mechanism (Brown et al., 1983) that controls the trafficking of kinase-negative mutants. These studies provide an unequivocal demonstration of internalization and altered trafficking of kinase-negative EGF receptors (Felder et al., 1990). These results are in agreement with the results of Glenney et al. (1988) and Chen et al. (1989) but in conflict with their interpretation. By applying a simple mathematical model (Wiley, 1988; Glenney et al., 1988; Chen et al., 1989) the lack of down-regulation of kinase-negative mutants was misinterpreted as an indication for reduced endocytosis rather than accelerated recycling (Felder et al., 1990). Cell Transformation and Cancer The importance of allosteric regulation of receptor activation and signal transduction is further emphasized by the fact that a large variety of structural alterations found in receptor-derived oncogene products lead to constitutive activation and, consequently, subversion of molecular control mechanisms and alteration of receptor signals. Thus, transforming receptor tyrosine kinase derivatives serve as valuable model systems not only for studying the mechanisms of oncogenesis but also for the analysis of structure-function relationships for these signal transmission molecules. Constitutive activation of receptor tyrosine kinase signaling functions can be achieved in a number of ways. For example, in the cases of v-e&B and v-kit, deletion of the extracellular binding domain eliminates the negative control that this structure normally exerts on the cytoplasmic domain. Even point mutations within the extracellular domain can lead to intracellular activation, as in the case of v-fms mutations at residues 301 and 374 (Woolford et al., 1988; Roussel et al., 1988). These mutations appear to induce and stabilize a conformational change equivalent to that triggered by ligand binding and possibly dimerization. Another dramatic effect of a single point mutation is exemplified by the Val+Glu conversion in the neu transmembrane domain (Bargmann et al., 1986) which suggests that this part of the putative receptor is involved in an overall conformational alteration that occurs upon interaction with the yet unidentified ligand. In this case, the transmembrane mutation results in constitutive receptor oligomerization (Weiner et al., 1989). Receptor-derived oncogenes possess other structural lesions such as cytoplasmic point mutations, deletions, and carboxy-terminal truncations that appear to enhance and modulate the transforming signal (Khazaie et al., 1988; Woolford et al., 1988). Activating receptor tyrosine kinase mutations seem to play a minor role in human cancer. The most common cellular lesion found in human cancers involves autocrine ac-

tivation in conjunction with receptor overexpression. Many tumors and tumor cell lines have been found to coexpress growth factors and their receptors, including TGFo, PDGFA, PDGF-B, acidic FGF, basic FGF, and their specific receptors. Thus, autocrine receptor activation represents yet another scenario of subversion of normal growth control. For mammary and ovarian carcinoma, extensive studies have demonstrated a direct correlation between the extent of overexpression of p185HER2/neu and a patient’s prognosis, a result that strongly suggests a critical role for this EGF receptor-like tyrosine kinase in tumor progression and perhaps even tumor initiation (Slamon et al., 1989). This possibility is further supported by efficient induction of mammary carcinoma in mice by an activated neu gene product (Muller et al., 1988) and transformation of NIH 3T3 cells by overexpression of unaltered p185HERz’neU (Hudziak et al., 1987). Analogous experiments with the EGF receptor indicated that autocrine stimulation of the overexpressed receptor was essential to achieve a transforming effect (Di Fiore et al., 1987; Velu et al., 1987; Riedel et al., 1988). In principle, every receptor with tyrosine kinase activity has oncogenic potential. One can anticipate that many more types of activating mutations, as well as specific instances of receptor tyrosine kinase overexpression, will be detected in animal and human tumors. The molecular identification and characterization of these mutants will not only provide important insights into fundamental mechanisms underlying receptor activation and normal growth control, but may also enhance our understanding of oncogenesis and open new avenues for diagnosis and therapy.

References Ballotti, R., Lammers, R.. Scimeca, I.-C., Dull, T., Schlessinger, J., UIIrich, A., and Van Obberghen, E. (1989). Intermolecular transphosphorylation between insulin receptors and EGF-insulin receptor chimerae. EMBO J. 8, 3303-3309. Bargmann, C. I., Hung, M. C., and Weinberg, R. A. (1988). Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of ~185. Cell 45, 649-657. Bertics, P. J.. Chen. W. S., Hubler, L., Lazar, C. S.. Rosenfeld, M. G., and Gill, G. N. (1988). Alteration of epidermal growth factor receptor activity by mutation of its primary carboxy-terminal site of self phosphorylation. J. Biol. Chem. 263, 3610-3617. Boni-Schnetzler, M.. and Pilch, P. F. (1987). Mechanism of EGFreceptor autophosphorylation and high affinity binding. Proc. Natl. Acad. Sci. USA 84, 7832-7836. Brown, M. S., Anderson, R. G. W., and Goldstein, J. B. (1983). Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell 32, 663-667. Chen, W. S., Lazar. C. S., Poenie, M., Tsren, R. Y., Gill, G. N., and Rosenfeld, M. G. (1987). Requrrement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. Nature 328, 820-823. Chen. W. S., Lazar, C. S., Lund, K. A., Welsh, J. B.. Chang, C-f?, Walton, G. M., Der, C. J., Wiley, H. S., Gill, G. N.. and Rosenfeld, M. G. (1989). Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulatron. Cell 59, 33-43. Chou, C. K.. Dull, T. J.. Russell, D. S., Gherzi, R.. Lebwohl, D., Ullrich, A., and Rosen, 0. M. (1987). Human insulin receptors mutated at the

Cell 210

ATP-binding sate lack protein tyrosine kmase activity and fail to mediate postreceptor effects of insulin. J. Biol. Chem. 262, 1842-1847. Cachet, C., Kashles, 0.. Chambaz, E. M., Borrello. I., King, C. R., and Schlessingec J. (1988). Demonstration of epidermal growth factorInduced receptor dimerization in lrvmg cells using a chemrcal covalent cross-linking agent. J. Biol. Chem. 263, 3290-3295. Czech, M. (1989). Signal transmission tors Cell 59, 235-238.

by the insulin-kke growth fac-

Davis, R. J. (1988) Independent mechanisms account for the regulation by protein kinase-C of the EGF-receptor affinity and tyrosineprotein kmase activity. J. Biof. Chem. 263, 9462-9469. DI Frore, l? P.. Pierce, J. H., Fleming, T. P., Hazan. R., Ullrich, A., King, C. Ft., Schlessinger, J., and Aaronson, S. A. (1987). Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell 57, 1063-1070. Downing, J. FT.,Margolis, B. L., Zilberstem, A., Ashmun. R. A., Ullnch, A , Sherr, C. J., and Schlessinger, J. (1989). Phospholipase C-r, a substrate for PDGF receptor kinase. is not phosphorylated on tyrosine residues during the mitogenic response to CSF-1. EMBO J. 8. 33453350. Escobedo, J. A., and Williams, L. T. (1988). A PDGF receptor domain essential for mitogenesrs but not for other responses to PDGF. Nature 335, 85-87. Felder, S., Miller, K., Moehren, G., Ullrich, A., Schlessinger, J., and Hopkins, C. A. (1990). Kinase activity controls the sorting of EGF receptor withm the multivesrcular body. Cell, in press. Glenney, J. R., Jr., Chen. W. S., Lazar, C. S.. Walton, G. M., Zokas, M. R., Rosenfeld, M. G., and Gill, G. N. (1988). Ligand-Induced endocytosis of the EGF receptor is blocked by mutational inactivation and by micromjection of anti-phosphotyrosine antibodies. Cell 52, 675684. Greenfield, C., Hils. I., Waterfield, M. D., Federwrsch, W., Wollmer, A., Blundell. T. L., and McDonald, N. (1989). EGF binding induces a conformahonal change in the external domain of its receptor. EMBO J. 8, 4115-4124. Haley, J. D.. Hsuan, J. J.. and Waterfield, M. D. (1989). Analysrs of mammalran fibroblast transformations by normal and mutated human EGF receptors. Oncogene 4. 273-283. Hammacher, A., Mellstrom, K., Heldin, C.-H., and Westermark, B. (1989). Isoform-specific induction of actm reorganization by plateletderrved growth factor suggests that the functionally active receptor is a drmer. EMBO J. 8, 2489-2495. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 247, 42-52. Hazan, R., Margolis. B.. Dombalagian, M.. Ullrich, A., Zrlberstein, A., and Schlessinger, J. (1990). identification of autophosphorylation sites of HER2/neu. Cell Growth Differ. I, 3-7. Heldin, C.-H., Ernlund, A., Rorsman, C., and Ronnstrand, L. (1989). Dimerization of the B-type platelet-derived growth factor receptors occurs after lrgand binding and is closely associated with receptor kinase activation. J. Biol. Chem. 264, 8905-8912. Honegger, A. M., Dull, T. J., Felder, S., Van Obberghen, E.. Bellot, F., Szapary, D., Schmidt, A., Ullrich, A., and Schlessinger, J. (1987a). Point mutation at the ATP binding site of EGF receptor abolishes protemtyrosine kinase actrvrty and alters cellular routing. Cell 51. 199-209. Honegger, A. M., Szapary. D., Schmidt, A., Lyall, R.. Van Obberghen, E.. Dull, T. J., Ullrich, A., and Schlessmger, J. (1987b). A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis. Mol. Cell. Biol. 7. 4568-4571.

Honegger, A. M., Kns. Ft. M., Ullrich, A, and Schlessinger, J. (1989). Evidence that autophosphorylation of solubilized EGF-receptors is mediated by mtermolecular cross phosphorylatron. Proc. Natl. Acad. SCI. USA 86, 925-929. Honegger, A. M., Schmrdt, A., Ullrich. A., and Schlessmger, J. (1990a). Evrcfence for EGF-induced intermolecular autophosphorylation of the EGF-receptor in livrng cells. Mol. Cell. Biol., in press. Honegger, A. M., Schmidt, A., Ullrich. A., and Schlessinger, J. (1990b). Separate endocytic pathways of kinase-defective and active EGFreceptor mutant expression in the same cells. J. Cell Biol., in press. Hudziak, pression formation USA 84,

R. M., Schlessmger, J., and Ullrich, A. (1987). Increased exof the putative growth factor receptor p185~~sn~causes transand tumongenesis of NIH 3T3 cells. Proc. Nat1 Acad. Sci. 7159-7163.

Kashles, O., Szapary, D., Bellot, F.. Ullrich, A., Schlessinger, J., and Schmidt, A. (1988). Ligandinduced strmulabon of EGF-receptor mutants wrth altered transmembrane regions. Proc. Natl. Acad. Sci USA 85, 9567-9571. Kazlauskas, A., and Cooper, J. A. (1989). Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins. Cell 58, 1121-1132. Khazaie, K., Dull, T. J., Graf, T., Schlessrnger, J., Ullrich, A., Beug, H., and Vennstrom, B. (1988). Truncation of the human EGF receptor leads to differential transforming potentials in primary avian frbroblasts and erythroblasts. EMBO J. 7, 3061-3071. Kraus, M. H., Issmg, W., Miki, T., Popescu, N. C., and Aaronson, S. A. (1989). Isolation and characterization of E/%3/33, a thrrd member of the ERBBlepidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc. Natl. Acad. Sci USA 86, 9193-9197. Kumjian, D. A., Wahl, M. I., Rhee. S. G., and Daniel, T. 0. (1989). Platelet-derived growth factor (PDGF) binding promotes physical association of PDGF receptor with phospholipase C. Proc. Natl. Acad. Sci. USA 86, 8232-8236. L’Allemarn, G., Seuwen, K., Velu, T., and Pouyssegur, J. (1989). Signal transduction in hamster fibroblasts overexpressing the human EGFreceptor. Growth Factors I, 311-321. Lammers, R.. Gray, A., Schlessinger, J., and Ullrich, A. (1989). Differential signalling potential of insulin- and IGF-l-receptor cytoplasmrc domains. EMBO J. 8, 1369-1375. Lax, I., Bellot, F., Hawk, R., Ullrich, A., Grvol, D., and Schlessinger, J. (1989). Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric human/chicken receptor molecules. EMBO J. 8. 421-427. Lee, J., Dull, T. J., Lax, I., Schlessinger, J., and Ullrich, A. (1989). HER2 cytoplasmic domain generates normal mitogenic and transformtng srgnals in a chimeric receptor. EMBO J. 8. 167-173. Lee, P. L.. Johnson, D. E., Cousens, L. S., Fried, V. A., and Williams, L. T. (1989). Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor. Science 245, 57-60. Lin, C. R., Chen, W. S., Lazar, C. W., Carpenter, C. D., Gill, G. N., Evans, R. M., and Rosenfeld, M. G. (1986). Protein kinase C phosphorylabon at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independent mechanisms. Cell 44, 839-848. Lrvneh, E., Prywes, R.. Kashles, O., Reiss. N., Sasson, I., Mary, Y., UIIrich, A., and Schlessmger, J. (1986). Reconstitution of human epidermal growth factor receptor and Its deletion mutants in cultured hamster cells. J. Biol. Chem. 261, 12490-12497. Livneh, E., Reiss. N.. Berent, E., Ullrich, A., and Schlessinger, J (1987). An msertional mutant of eprdermal growth factor receptor allows dissectron of diverse receptor functions. EMBO J. 6, 2669-2676.

Honegger, A., Dull, T. J., Bellot, F.. Van Obberghen. E., Szapary. D.. Schmidt, A.. Ullrich, A., and Schlessinger, J. (1988a). Biological acbvitres of EGF-receptor mutants with individually altered autophosphorylation sites. EMBO J. 7, 3045-3052.

Livneh, E., Dull, T., Prywes. R., Ullrich. A., and Schlessinger, J. (1988). Release of phorbol ester-induced mitogenic block by mutation at Thr654 of epidermal growth factor receptor. Mr$. Cell. Biol. 8, 2302-2308.

Honegger, A., Dull, T. J., Szapary, D., Komoriya. A., Kris. R.. Ullrich, A.. and Schlessmger, J. (1988b). Kmetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with indivtdually altered autophosphorylation sites. EMBO J. 7. 3053-3060.

Lopez-Rivas. A., Mendoza, S. A., Nhberg, E., Sinnett-Smith. J., and Rozengurt. E. (1987). Caa+-mobilizing actrons of platelet-derived growth factor differ from those of bombesin and vasopressm In Swiss 3T3 mouse cells. Proc. Natl. Acad. Sm. USA 84. 5768-5772.

Review: Receptor Tyrosine Kinases 211

Margolis, B., Lax, I., Kris. R.. Dombalagian, M., Honegger, A. M., Howk, R., Givol, D., Ullrich, A., and Schlessinger, J. (1989a). All autophosphorylatron sites of epidermal growth factor (EGF) receptor and HER2lneu are located in their carboxy-terminal tails: identification of a novel site In EGF receptor. J. Biol. Chem. 264, 10667-10671. Margolis, B., Rhee, S. G., Felder, S., Mervic, M., Lyall. R., Levitzki, A., Ullrich, A., Zilberstein, A., and Schlessinger, J. (1989b). EGF Induces phosphorylation of phospholipase C-II: a potential mechantsm for EGF receptor srgnaling. Cell 57; 1101-1107. Margolis, B., Bellot, F., Honegger, A. M., Ullrich, A., Schlessinger, J.. and Zilberstern, A. (1990a). Tyrosine kinase activity is essential for the association of phospholipase C-7 with EGF-receptor. Mol. Cell. Biol. 70, 435-441. Margolis, B.. Zilberstein, A., Franks, C., Felder, S., Kreamer, S., Ullrich. A., Rhee, S. G., Skoreckr, K., and Schlessinger, J. (1990b). Phospholrpase C-y overexpression enhances PDGF-induced IPs formation but not calcrum signaling and mitogenesis. Science, in press.

Sherr, C. J. (1987). Transforming potentral of the c-fms proto-oncogene (CSF-1 receptor). Nature 325, 549-552. Roussel, M. F., Downing, J. R.. Rettenmier, C. W., and Sherr, C. J. (1988). A point mutation in the extracellular domain of the human CSF1 receptor (c-fms proto-oncogene product) activates its transforming potential. Cell 55, 979-988. Russell, D. S., Gherzi, R., Johnson, E. L., Chou, C.-K., and Rosen, 0. M. (1987). The protein-tyrosine kinase activity of the insulin receptor IS necessary for insulin-mediated receptor down-regulatron. J. Biol. Chem. 262, 11833-11840. Ruta, M., Burgess, W., Givol, Crumley, G., Dionne, C., Jaye, tor for acidic fibroblast growth encoded by the fms-like gene 8722-8726.

D., Epstein, J.. Neiger, N., Kaplow, J., M.. and Schlessinger, J. (1989). Recepfactor is related to the tyrosine kinase (FLG). Proc. Natl. Acad. Sci. USA 86,

Schlessingec J. (1988). Signal transduction by allosteric receptor olrgomenzation. Trends Biochem. Sci. 13, 443-447. R. A., Hart, C. E., Phillips, P. E , Forstrom, J. W., Ross, R., M. J., and Bowen-Pope, D. F. (1989). Two different subunits asto create isoform-specific platelet-derived growth factor recepBiol. Chem. 264, 8771-8778.

McClain, D. A., Maegawa, H., Lee, J., Dull, T, J., Ullrrch, A., and Olefsky, J. M. (1987). A mutant insulin receptor with defective tyrosine kinase drsplays no biologic activrty and does not undergo endocytosis. J. Biol. Chem. 262, 14663-14671.

Seifert, Murray, sociate tors. J.

Meisenhelder, J., Sub, P-G., Rhee, S. G., and Hunter, T. (1989). Phospholipase C-y is a substrate for the PDGF and EGF receptor proterntyrosine kinases in vivo and in vitro. Cell 57, 1109-1122.

Shrer, P., and Watt, V. M. (1989). Primary structure of a putative receptor for a ligand of the Insulin family. J. Biol. Chem. 264, 14605-14608.

Molloy, C. J., Bottaro, D. P., Fleming, T. P, Marshall, M. S., Gibbs, J. B., and Aaronson, S. A. (1989). PDGF Induction of tyrosine phosphorylation of GTPase activating protein. Nature 342, 711-714. Moolenaar, W. H., Bierman, A. J., Tilly, B. C., Verlaan, I., Defize, L. H. K., Honegger, A. M., Ullrich, A., and Schlessinger, J. (1988). A pomt mutation at the ATP-binding site of the EGF-receptor abolishes signal transductron. EMBO J. 7. 707-710. Morrison, D. K., Kaplan, D. R., Escobedo, J. A., Rapp, U. R., Roberts, T M., and Williams, L. T. (1989). Drrect actrvation of the serinelthreonine kmase activity of Raf-1 through tyrosine phosphorylation by the PDGF 8-receptor. Cell 58, 649-657. Muller, W. J., Sinn, E.. Pattengale, P K.. Wallace, R., and Leder, P (1988). Single-step rnductron of mammary adenocarcinoma in transgenie mice bearing the activated c-neu oncogene. Cell 54, 105-109. Nrshibe, S., Wahl, M. I., Wedegaertner, P. B., Kim, J. J., Rhee, S. G., and Carpenter, G. (1990). Selectivity of phospholipase C phosphorylatron by the eprdermal growth factor receptor, the insulin receptor, and their cytoplasmic domains. Proc. Natl. Acad. Sci. USA 87, 424-428. Nishizuka, Y. (1988). The molecular heterogeneity of protein kinase C and its rmplications for cellular regulation. Nature 334, 661-668. Northwood, I. C., and Davis, R. J. (1988). Activation of the epidermal growth factor receptor tyrosine protein krnase in the absence of receptor oligomerrzation. J. Biol. Chem. 263, 7450-7453. Pasquale, E. B., and Singer, S. J. (1989). ldentrficatron of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library. Proc. Natl. Acad. Sci. USA 86, 5449-5453. Prywes, R., Livneh, E., Ullrich. A., and Schlessinger, J. (1986). Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization. EMBO J. 5, 2179-2190. Rredel, H., Dull, T. J., Schlessinger, J., and Ullrich, A. (1986). A chimaeric receptor allows insulin to stimulate tyrosine krnase activity of epidermal growth factor receptor. Nature 324, 68-70. Riedel, H., Massoglia, S., Schlessinger, J., and Ullrich, A. (1988). Ligand actrvation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblsts. Proc. Natl. Acad. Sci. USA 85, 1477-1481.

Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G.. Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A., and Press, M. F. (1989). Studies of the HER-2/neu proto-oncogene in human breast and ovanan cancer. Science 244, 707-712. Soos, M. A., and Saddle, K. (1989). lmmunologrcal relationshrps between receptors for insulin and insulrn-like growth factor I. Biochem. J. 263, 553-563. Stern, D. F., Kamps, M. P., and Lao, H. (1988). Oncogenic activation of ~185”~~ strmulates tyrosine phosphorylation in viva. Mol. Cell. Biol. 8, 3969-3973. Taylor, G. R., Reedjik. M., Rothwell, V. R., Rohrschneider, L. R., and Pawson, T. (1989). The unique insert of cellular and viral fms protein tyrosine-kinase domains is dispensable for enzymatic and transforming activities. EMBO J. 8, 2029-2037. Tornqvist, H. E.. and Avruch, J. (1988). Relationship of site-specific 8 subunit tyrosrne autophosphorylation to insulin activation of the insulin receptor (tyrosrne) protein kinase activity. J. Biol. Chem. 263, 45934601. Varticovski, L., Druker, 8.. Morrison, D., Cantley, L., and Roberts, T. (1989). The colony stimulating factor-l receptor associates with and activates phosphatidylinositof-3 kinase. Nature 342, 699-702. Velu. T. J., Begumot, L., Vass, W. C., Willingham, M. C., Merlino, G. T., Pastan, I , and Lowy, D. R. (1987). Epidermal growth factor-dependent transformatron by a human EGF receptor proto-oncogene. Science 237, 1408-1410. Velu. T. J., Vass, W. C.. Lowy, D. R., and Beguinot, L. (1989) Functional heterogeneity of proto-oncogene tyrosine krnases: the C termtnus of the human epidermal growth factor receptor facrlitates cell proliferatron Mol. Cell. 6101. 9, 1772-1778. Wahl, M., Nishibe, S., Suh, P-G., Rhee, S. G., and Carpenter, G. (1989). Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium. Proc. Natl. Acad. Sci. USA 86, 1568-1572. Wedekind, F.. Baer-Pontzen. K., Bala-Mohan, S., Choli, D., Zahn, H., and Brandenburg, D. (1989). Hormone brnding site of the insulm receptor: analysrs using photoaffinity-mediated avrdm complexing. Biol. Chem. Hoppe-Seyler 370, 251-258. Weiner, D. B., LIU, J., Cohen, J. A., Williams, W. V., and Greene, M. (1989). A point mutation in the neu oncogene mimics ligand Induction of receptor aggregation. Nature 339, 230-231.

Riedel, H., Dull, T. J., Honegger, A. M.. Schlessinger, J., and Ullrich, A. (1989). Cytoplasmic domarns determine signal specificity, cellular routing characterrstics and Influence ligand bindrng of epidermal growth factor and insulin receptors. EM60 J. 8, 2943-2954. Rosen, 0. M., Herrera, R., Olowe, Y,, Petruzzelli, L. M., and Cobb, M. H. (1983). Phosphorylatron actrvates the insulrn receptor tyrosrne kinase. Proc. Natl. Acad. Sci. USA 80, 3237-3240.

White, M. F., Livingston, J. N.. Backer, J. M., Lauris, V., Dull, T. J., UIIrtch, A., and Kahn, C. R. (1988). Mutation of the Insulin receptor at tyrosme 960 inhrbrts signal transmission but does not affect its tyrosine kinase actrvrty. Cell 54, 641-649

Roussel, M. F., Dull, T J.. Rettenmier, C. W., Ralph, P, Ullrich. A.. and

Wrley, H. S. (1988). Anomalous

bindmg of epidermal growth factor to

Cell 212

A431 cells IS due to the effect of high receptor densities and a saturable endocytrc system. J. Cell Biol. 107, 801-810. Wrllrams, L. T. (1989). Srgnal transduction by the platelet-dewed growth factor receptor. Science 243, 1564-1570. Wrttbrodt. J., Adam, D.. Malitschek, B.. Maueler, B., Raulf, F., Telling, A.. Robertson, S. M., and Schartl, M. (1989). Novel putative receptor tyrosme kinase encoded by the melanoma-Inducing Tu locus in Xrphophorus. Nature 347, 415-421. Woolford, J. W.. McAulrffe, A., and Rohrschneider, L. R. (1988). Activation of the felme c-fms proto-oncogene’ multrple alterations are required to generate a fully transformed phenotype. Cell 55, 965-977 Yarden, Y., and Schlessinger. J. (1985). EGF receptor self-phosphorylatron is medated by an intermolecular allosteric process. In Growth Factors rn Biology and Medicme, (London: Pitman), pp. 23-45. Yarden, Y.. and Schlessinger, J. (1987a). Self-phosphorylahon of eprdermal growth factor receptor: ewdence for a model of mtermolecular allosteric activation. Biochemistry 26, 1434-1442. Yarden, Y., and Schlessinger, J. (1987b). Epidermal growth factor induces rapid, reversible aggregatron of the purified epidermal growth factor receptor. Biochemistry 26. 1443-1451. Yarden, Y., and Ullrich, A. (1988). Growth factor receptor tyrosme krnases. Annu. Rev. Biochem. 57, 443-478.