Bioch#nica et Biophysica Acta. I(D5 (lOll) 127-139 © 199t Elsevier Science Publishers B.V. All rights reserved 0167-4889/91/$03.50 ,4DONIS 0167488qgI00273Q

127

Minireview

BBAMCR 13042

Molecular biology of a-adrenergic receptors: implications for receptor classification and for structure-function relationships Jon W. I~masney, Susanna Cotecchia, Robert J. gefkowitzand Marc G. Caron Departments of Pathology, Medicine, Biochemistry, Cell Biology and the Howard Hnghes Medical Institute. .Duke University Medical Center, Durham. iV(" (U.S.A.) {Received 26 June 199I)

Key words: O protein; Adrenergic rcccpt~)r: Catccholamiac

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

127

II. at-Adrenergic receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Elucidation of ~he primary structure of distinct al-adrencrgic receptor subtypes . . . . . . . . . . . B. Tissue distribution of the a radrenergic receptor subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . C. Gene organization and chromosomal localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I~ Signal transductlon mechanisms of at-adrenergic receptor subtypes . . . . . . . . . . . . . . . . . . . . F. Structure/functlon of aradrenergic receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

128 129 [2q 130 130 132 133

Ill. a2-Adrenergic receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Elucidation of the primary structure of a2-ad~energi¢ receptors . . . . . . . . . . . . . . . . . . . . . . . B. Tissue distribution of the cloned av-adrenergic receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Gene organization and chr'~mosoraal localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Structure of a2-adrenergie receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Signal transductlon of az-ad-~',~ergi¢ receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

133 134 134 125 135 136

IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

137

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

137

!. Introduction

regulatory proteins or G-proteins. G-protein coupled r e c e p t o r s are i m p o r t a n t physiologic r e g u l a t o r s b e c a u s e

The a-adrenergic receptors are members of the large and diverse family of integral membrane proteins which mediate effects via guanine nucleotide-binding

Abbreviations: G protein, guanine nucleotide-binding regulatory protein; AR, adl~nergic receptor; MSD, transmembrane spanning domain; PKA, protein kinase A; PI, lmlyphosphoinositide; IP3, inositol |,4,5.trisphosphate; PA2, - l o g Kou where KDn = dissociation conslant for receptor-antagonist interactions; ?LC, phospholipas¢ C; kb, kilobase; Ki, dissociation constant; bp, base pair; 51-1TIA. 5-hydraxytryptamine (serotonln) receptor; [12~i]HEAT, [t2Sl]iodo-2[~(4-hydroxyphenyl)ethylaminomethyl]tetralone. Correspondence: J.W. Lomasacy, Department of PathologY, Duke University Medical Center, Durham, NC 27710, U.S.A.

they are the target for a multitude of hormones, neurotransmitters as well as autocrine and paracrine factors, It has recently become apparent that though there is a great number of primary messengers, there is an even greater number of distinct receptors that elicit the specific cellular responses [l], This realization has come about in large part through the molecular cloning of a large number of eDNA/genes encoding for these receptors, some of which were not previously identified by radioligand binding and functional studies. The adrenergic receptor family serves as an example of this diversity of receptor subtypes; nine distinct receptors mediate the varied effects of only two physiological agonists, epinephrine and norepinephrine [2-11]. Though members of this G-protein coupled receptor

t28 supeffamity bind various types of ligand and mediate a wide variety of responses, the primary structure of these proteins is remarkably similar. Perhaps most characteristic is the presence of seven stretches of hydrophobic amino acid residues which are thought to span the plasma membrane [12-14]. In addition to the seven transmembrane spanning domains, the topographic model for these receptors predicts that the amino-terminus and three hydrophilic loops extend into the extra-cellular space, and that the remaining three loops and carboxTl-terminus extend into the intracellular space. This topography has been most rigorously proven for bacteriorhodopsin, where isolation of relatively large amounts of pure protein have allowed detailed electron diffraction and low-dose electron microscopic studies of the tertiary structure [15-17]. Extensive biochemical and physical data also support this model for the visual protein rhodopsin [18]. Support of this same topography for the adrenergic receptors includes extensive similarity of the primary protein structures to bacteriorhodopsin and rhodopsin as well as biochemical data from studies of the /32-adrcnergic receptor using limited proteolysis ,and antipeptide antibodies [19,20]. The focus of much investigation has centered on determining the molecular mechanisms by which adrenergic receptors and other G-protein coupled receptors elicit specific and varied cellular responses by binding ligands and activating G-proteins. The knowledge of the corresponding nucleotide sequence not only gives more detailed information about the structure of the receptor protein, but also allows for manipulation of this structure by changing the nucleotide sequence of the eDNA. Studies of such mutant receptors has increased dramatically our knowledge about the structural domains of the receptor which are involved in ligand binding and G-protein activation [21]. The g-adrenergic receptor is among the most studied of the G-protein coupled receptors in this regard, and serves as a model for all the adrenergie receptors. An obvious yet difficult question arising from the recent proliferation of adrenergic receptors is: what role does the diversity of subtypes have in mediating the varied yet specific ceUuiar effects of the catecholamine agonists? Again the isolation of the receptor eDNA/genes has been extremely important by facilitating the mapping of the tissue Ioealizations of the different adrenergic receptor subtypes. It is quite lmssible that the different effects elicited by a single hormone in different tissues may be due in large part to the differential expression of receptor subtypes in those tissues. Conversely, there is also evidence for the ability of a single receptor to couple to more than one second messenger pathway and hence be ~tentially able to mediate different effects depending on hormone concentration or coexpression of various differ-

ent G-proteins and effector molecules. The isolation of the eDNA/genes has also allowed for the careful investigation of the ligand binding properties of the different subtypes. The expression of a single receptor in transient or permanent cell lines, to levels much higher than those obtained in rive has made it possible to discover pharmacologic agents which may discriminate between various receptor subtypes and has facilitated the study of the signal transduct~on mechanisms of each receptor. This review will attempt to summarize recent information about the subfamily of a-adrenergie receptors gained mostly as a direct result of the cloning of the eDNA/genes. 11. a j-Adrenergic receptors

al-Adrenergic receptors (AR) are present in many tissues including brain, heart, smooth muscle, liver, spleen and presynaptic nerve terminals, where they mediate a variety of effects including vasoconstriction, glycogenolysis, cardiac inotropy and chronotropy. Recent pharmacological and functional studies have suggested that some of these responses may be mediated by distinct a1-AR subtypes. Radioligand binding studies have shown heterogeneity of al-adrenergie receptors by demonstrating two classes of neAR binding site with various antagonists and agonists. Morrow and Creese were among the very first groups to report heterogeneity of aradrenergic receptors. They demonstrated biphasic displacement isotherms at al-adrenergic binding sites in rat cerebral cortex with the antagonists W~4101 and phentolamine [22]. They suggested that the high affinity site in this tissue be termed the a,A-AR, while the low affinity site be termed the am. Subsequent reports have not only confirmed these results but have added to the list of compounds that discriminate between oq-ARs. Ox~metazoline, 5-methyl-urapidil, niguldipine, methoxamine, amidephrine and benoxathian have been reported to have high affinity for the atn subtype, while spi~rone, the classical D2 dopamine receptor antagonist, is the only drug reported to date to have relatively higher affinity for the oqB subtype [23-26]. In addition, the alkylating agent chloroethylclonidine (CEC) while having equal apparent affinity at both at-AR, has been shown to irreversibly inactivate only the %,~ subtype [27-29]. It should be pointed out that while these compounds apparently discriminate between only two a~-AR in the various tissues examined, the existence of additional at-AR has not been discounted. Therefore, for the purposes of classification the a~-AR in rat cerebral cortex with high affinity for WB4101 should be designated as the ate, as originally suggested by Morrow and Creese, while the low affinity site should be designated as the alB.

129 II-A. Elucidation o f the primary structure o f distinct a fadrenergic receptor subtypes

Molecular cloning studies have identified not only the two distinct subtypes of at-adrenergic receptor postulated on the basis of pharmacological characteristics but also a third novel subtype not previously identified. The eDNA encoding for the a]-adrenergic receptor expressed in the Syrian hamster smooth muscle cell line DDT1 MF-2 was isolated using oligonucleotide probes designed from partial amino acid sequence of the purified receptor [9]. Two other distinct at-AR cDNAs were subsequently isolated from bovine brain and rat brain eDNA libraries, using fragments of the hamster at-adrenergic receptor eDNA as probes [10,11]. The receptor isolated from the hamster cell line DDTI MF-2 appears to have the characteristics of the pharmacologically defined a,a subtype, while the receptor cloned from rat brain appears to represent the atA subtype. The receptor from bovine brain, while having some ligand binding characteristics similar to the atA subtype represents a third distinct receptor designated as the a~c adrenergie receptor. The ligand binding properties of the three at-adrenergic receptors after expression in COS-7 cells is shown in Table I. It is apparent that the three receptors display unique pharmacologies. The rat otlA receptor is distinguished from both the hamster ata and bovine ate receptors by having at least a 104old higher affinity for the agonists NE, EPI and phenylephrine. It is further distinguished from the aia subtype by its higher affinity for the agonist methoxamine and the antagonist WIM101; and from the ate subtype by oxymetazoline and phentolamine, The ata and ate subtypes are

distinguished from each other by methoxamine, oxymetazoline, phentelamine and WB4101. Sensitivity to irreversible inactivation by the alkylator CEC has been used by several groups to distinguish between the a,A and 0qa adrenergic receptors. Results of studies in which COS-7 cell membranes transfected with each of the three at-AR subtypes were exposed to 10 or 1(30 ~.M CEC, have shown that the a]A subtype appears to be less sensitive to inactivation by l0/.~M CEC (15%) than the a,a (70%) and ate (80%) subtypes [1 I]. I1-B. Tissue distribution o f the aradrenergic receptor subtypes

Northern blot analysis (Table II) has demonstrated unique tissue distributions for each of the three subtypes. In the rat the a n subtype is expressed in the tissues liver > heart > cerebral cortex > spleen [I!]. On the other hand, the ajn receptor is most abundant in vas deferens, followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. This assessment of the tissue distributions of these receptors is in agreement with their distributions as determined by WB4101 competition curves and CEC inactivation studies [22,29t. Quite unexpectedly, expression of the ate receptor was not detected in any rat tissues by Northern analysis. To date the expression of this receptor has been detected only in rabbit liver and in the granular cell layer of the human dentate g/rus (an exhaustive survey of the human brain has not been performed) [10,30]. This suggests that the expression of this receptor is highly selective and limited, These findings also serve as an example of the fact that

TABLE l Competition by agon~s and antagonists.for the bindingof [ tZSl]HEAT to the three cloned a t-adrenergicreceptorsubtypes, and ['~H]yohimbineto the three a2.adrtnergic receptorsitbtypes Ki expressed as nM. n.d. = not determined. Values taken from Refs. 6, 7, 9, 10, tl,

Agonists (-)Epinephrine

(-)Norepinephine ( + )Epinephrine Methoxamine Phenylephrine Ox~metazoline p-Aminocloaidine Antagonists Prazosln Phentolamine lndoramine Corynanthine WB41Ol Rauwolscine SI'¢.Ft040"/8

Rat a,A

Rat a,B

Bovineale

Human a2C2

Human a2C4

Human azCI0

546 100 18100 110000 ! 440 2140 n.d.

4690 10500 32000 1610000 23900 842 680

6250 9730 43000 203000 47800 114 59o

1850 1260 8420 n.d. [].d. 1500 ] 20

318 606 n.d. n.d. 2900 I~ 97

1670 3680 n.d. n.d. 1500 13.2 3I

0.33 11I 61l ~3 2.1 n.d, n.d.

0.56 340 226 517 28.6 3200 n.d.

0.37 15 12 142 0.68 t400 n.d.

293 9.2 n.d. 1002 132 I1 132

68 14.4 n.d182 13 2.1 13

2240 6.2 n.d. 1190 47 7.1 47

130 T A B L E II

a.Adrenergic receptor subtype mRNA disrritn~tion alg

message size: 2.4 kb

atA 3.0 kb

ate

a:Cl0 3.8 kb

a2C2 4,1 kb

a2C4 2.9 kb

o + + +

o o

+ + ~- + +

o o

o + + +

pituitaw hippocampus

o +

brain stem

+ + +

+ +

o

+ + + +

o

+ + +

cerebellum

+ + +++ + + + + + + a + + o

+ +++ + o +

o

o

+ + +

o o ob o o

+ + +-6+ + + o +

+ +

o 4- + + + o o

+ + + +++ o O o o

-~ ÷ + + o + o 0 n.d. n.d. n.d. n.d,

+ + + + + + + + + + n.d. n.d. n.d. n.d,

o o o o 0 n.d. n.d. n.d. n.d.

o + + + n.d. n,d. + + + + o 0 o

o o o n.d. n.d. o o o c o

o o o rod. n,d. o + 4o + +

cortex kidney

liver lung aorta

heart skel. muscle

spleen adip. tissue vas deferens HT29 NGI0S-IS neonatal lung OK

a Liver + 3 . 3 kb. m R N A was detected in rabbit liver. e p R N G rat kidney a , - a d r e n e r g i ¢ receptor. + + + + = very strong signal; o = no signal d e t e c t e d ; n.d. = not

determined.

different species may preferentially express different receptor subtypes in tissues; e.g., the ate receptor is expressed in rabbit liver, while the a,,~ subtype is

human chromosome 5, while the gene encoding for the ate resides on human chromosome g. In situ hybridization of cDNA and genomie probes to human metaphase

expressed in rat liver. Important features of the three al-AR subtypes are summarized in Table IlL

chromosomes has further localized the atB-AR gene to region 5q32-34 and the alA-AR gene to 5q23-31 [31],

H-C. Gene organization and chromosomal localization

The gene for the/~z-AR is also located in this region 5q31-33. The close proximity of the three adrenergic receptor genes on the same chromosome suggests that

In contrast to the genes for the three ,8 and three a2

adrenergic receptor genes isolated to date, the genes encoding for the atB and ale adrenergic receptors contain at least one intron. It has not been determined whether or not the alA-AR gene contains introns.

this family of proteins may have arisen at least in part by gene duplication.

II.D, Protein structure

Hybridization of the eDNA or portion of the gene with chinese hamster-human somatic cell hybrids shows that the genes for the a,A and am-AR are located on

Hydropathy analysis of the deduced amino acid sequences indicate that there are seven hydrophobie clusters of 20-25 residues, each separated by stretches

TABLE ili

a j -,4drenergic receptor classiftcation Subtype H u m a n chromosome

alA 5

Asonlsts

NE >

Antagonists R a t tissue localization

Epi

¢~1B 5

ale 8

Oxy > Epi 2:

Oxy :*, E p i >

Phenyl > Oxy

NE > Phenyl

NE > Phenyl

Pra > WB4101 :a> Phent cerebral cortex

Pra :~ W B 4 1 0 I > Phent c e r e b r a l cortex

P r a 2: W B 4 1 0 I > rabbit liver

hippocampus

liver

vas deferens

heart

aorta

Glycosylalion sites

yes

A m i n o acids

560 rat (11)

yes 515 hamster (9) 515 rat (54)

yes 466 bovine riO)

Phent

131

of hydrophilic residues; a pattern characteristic of all G-protein coupled receptors isolated to date [32]. These hydrophobic transmembrane spanning domains (MSD) are the most conserved regiops of the different members of the G-proteln coupled receptors. The aminoterminus, carboxyl-terminus and third cytoplasmic loop are the regions which differ most in length and amino acid composition. Since all adrenergic receptors bind the same endogenous ligands (epinephrine, norepinephrine), it has been postulated that the conserved MSD may be involved in ligand binding while the more divergent domains, especially the third cytoplasmic loop, may mediate coupling to the heterogeneous Gproteins which couple to varied signalling pathways. In fact, analysis of adrenergic and other G-protein coupled receptors whose primary structures have been modified by either deletion, insertion or exchange of various receptor domains has indicated that the MSD do make up the ligand binding site while the third intracellular loop and portions of the carboxyl-termintzs predominately mediate G-protein interactions [33-37].

Among adrenergic receptors, amino acid identities in the MSD range from 36 to 73%. Between members of the same subtype group (atB, alA, or ale), the MSD identity is usually 70-80%, while between different subtype groups (a, vs. otz, a 2 vs. fl etc.) the MSD identities are characteristically lower 30-40%. The deduced amino acid sequence and putative topography of the hamster azB adrenergic receptor is depicted in Fig. 1. The amino acid identities with residues in the MSD of other adrenergie receptors is as follows: rat ata 73%, bovine ale 65%, hamster .82 45%, and rat a2B 45% [11,10,38,8]. The deduced amino acid sequences of the three cloned al-adrenergic receptors range from 466 to 560 amino acids. From the primary structures the predicted molecular weights range from 37280 to 44800. The size of the ~xta-adrenergic receptor as determined bioebemicalfy from both purified receptor and crude receptor preparations identified by photoaffinity labeling is (Mr=80000) [39,40]. The observed size of the receptor is larger than the predicted size mostly as a result of posttrans-

EBTBBCEL

L UL B R

INTBBCELLULBB

tef~n~ Fig, l. Seven transmembtane-spanningmodel of the han~ter aza-adrenergicreceptor, Solio circles indicate amino acid identicalto the correspondingpositionin the hamster~2*adrenerg~creceptor, Transmembranedomainsare definedby hydropathicityanalysis[32]. Potential sitesof N-linkedgtycosylationare indicatedbycrosses.

132 lational modification of the receptor. Such modifications include the addition of complex type N-linked oligosaccharides to asparagine residues present in the the amino-terminus of the receptor. The Oqa adrenergic receptor purified from liver and rat brain has previously been shown to contain N-linked oligosaccharides [41]. After removal of N-linked carbohydrates by peptide-N4(n-acetyl-B-glucosaminyl)asparagine amidase, the am-adrenergic receptor from DDTI MF-2 cells has an Mr = 50000-55000 [41]. Similar biochemical information is lacking on the alA and a,c subtypes. Thus, the predicted and observed sizes of the peptide backbone of the am-adrenergic receptor are consistant with each other. Other structural features of the three at adrenergie receptors include the presence of a potential site for palmitoylation in the carboxyl-terminus. The G-protein coupled receptor rhodopsin has been shown to be aeylated by palmitic acid at two cysteine residues in the carboxyl-terminal tail [42]. One of these residues is conserved in an equivalent position in most members of the G-protein coupled receptor family. This residue CYS341 of the l/2 adrenergic receptor carboxy terminus is also covalently modified by thioesterification with palmitic acid [43]. It is not known whether the conserved cysteine present in the three at-adrcnergic receptors is also palmitoylated. The third cytoplasmic loop of the at-adrenergie receptors is intermediate in length compared with that of the /3 (short) or otz (long), while the carboxylterminus is the longest among the adrenergic receptors. Several serine and threonine residues present in the intracellular loops and in the carboxyl-terminus are potential sites for protein kinase C phosphorylation. In vitro phosphorylation of the ala adrenergic receptor by protein kinase C and A has been reported and may represent a mechanism for receptor regulation in vivo [44-46]. Interestingly, the fflA receptor lacks a consensus site for protein kinase A (PKA) phosphorylation, while both the ata and alc-adrenergic receptors contain a conserved PKA site in the third cytoplasmic loop. This suggests that mechanisms of a,A-adrenergic receptor regulation may be different from those regulating the a,B and ale subtypes.

II-E. Signal transduction mechanisms of c~Fadrenergic receptor subtypes Activation of at-adrenergic receptors causes polyphosphoinositide (Pl) hydrolysis catalyzed by phosphollpase C in almost all tissues where this effect has been examined. The resulting products of PI hydrolysis, including IP3 and diacylglycerol, increase intracellular calcium and activate protein kinase C, respectively {47]. However, functional heterogeneity of aFadrenergic receptor-mediated responses has been described in sev-

eral studies (for review see Ref. 48). A wide range of differences in pA 2 * values for tr-adrenergic receptor antagonists have been reported, as well as differences in agonist potency series for acadrenergic receptormediated vasoconstriction in various species. In addition, ~h-adrenergic receptor-mediated constriction of different smooth muscles shows a marked variation in the requirement for extraceUular calcium [28]. Recent studies have shown that other signalling pathways such as phosphatidylcholine specific phospholipase D [49] and phospholipase A 2 [50] can also be activated by a~-adrenergic receptor stimulation. These results have strongly suggested the existence of different a,-AR subtypes with distinct functional properties. However, the comparison among at-adrenergic receptor mediated responses in different tissues has not allowed any conclusive assessment of functional differences existing among aj-adrenergie receptor subtypes. In fact, the differences observed in a~-adrenergic receptor-mediated effects in various tissues or cell types could be determined by several factors other than the presence of distinct receptor subtypes. These factors include variations in receptor number and differences in the composition of G proteins and/or effector molecules. The factors acting individually or in concert might ultimately result in different patterns of receptor coupling.The expression of the individual genes of different receptor subtypes in a variety of cell lines represents the most accurate tool to directly assess the functional properties of distinct receptor molecules. This approach has been used to study the signal transduction mechanisms of the three acadrenergic receptor subtypes recently cloned. To date no dramatic differences have been observed between the three receptors. All three at-adrenergic receptors (ala, a~A and a,c) are able to stimulate PI hydrolysis via a PTX-insensitive G-protein, when expressed in COS7 cells (Ref. 30 and Lomasncy, J.W., Schwinn, D.A., Cotecchia, S., Caron, M.G. and Lefkowitz, R.J., unpublished data). Though the receptors appear to have similar signal transduction mechanisms, several differences may exist. Possible clues to significant differences may reside for example :,, the fact that even though the agonist potency for the ate is comparable to that of the a m, the ale.mediated release of inositol phosphates is 2-3-fold greater than the ale both in COS7 and Hela cells [30]. This might indicate that the alc-adrenergic receptor is coupled more efficiently to PLC than the am-adrenergic receptor. Obviously, further studies will be necessary to obtain a more detailed characterization of the signalling properties of these

* p.42 ffi- log gnu = dissociationconstant for receptor-antagonist interactions.Cell SurfaceReceptors:A Short Courseon Theory and Methods,by Lee E. Limbird,MartinusNijhoff,1986.

133 a~-adrenergic receptor subtypes. These studies might illuminate some of the findings previousIy obtained by functional studies in different tissues. II-F. Structure~function of a t-adrenergic receptors

In order to identify the regions of the a ~-adrenergic receptor involved in its coupling to PLC, a variety of chimeric and site-directed mutant receptors have been constructed [51]. A chimeric 132/a t receptor in which the entire third intracellular loop of the/32-adrenergic receptor was replaced with the corresponding region of the am-adrenergic receptor was able to stimulate inositol phosphate release in the presence of catecholamines. This result indicates that the putative third intracellular loop of the a~-adrenergic receptor confers to the /32-adrenergic receptor the ability to stimulate PLC. Recently, we have observed that the property of coupling to PLC via an as of yet unidentified Ga subunit resides in a region as small as 27 amino acid (residues 233-259) in the N-terminal portion of the third loop of the a m [52]. This 27 amino acid region of the aln is homologous to the corresponding region of the atA having 62% amino acid conservation and is 55% conserved with that of the a~c-adrenergic receptor. This striking conservation of structure in a region of the receptor which otherwise is not conserved, suggests an important role of this sequence as a recognition site for the G-proteins coupled to the al-adrenergic receptor family. While the sequences involved in receptor-G-protein interaction seem to reside in the N-terminal portion of the third loop, the C-terminal region of the third loop of the a~-adrenergic receptor also plays a crucial role for productive coupling of the receptor to PLC. In fact, single amino acid substitutions (Ala-293 ~ Leu, Lys290 ~ His) in the C-terminus portion of the third loop increased the affinity of agonist binding to the atr~adrenergie receptor and its potency to activate PLC by 1-2 orders of magnitude [53]. Cells expressing single point mutants had increased basal levels of ino'sitol phosphates in the absence of added agonist, suggesting that these mutant receptors may be constitutively active. The regions of the at-adrenergic receptor involved in receptor coupling to PLC are similar to those previously shown to be involved in coupling of the fl2-adrenergie receptor to adenylyl cyclase [34,36], indicating several generalities in the structure-function relationship of the G-protein-coupled receptors.

111. a2-Adrenergic receptors a2-Adrenergic receptors are present in a variety of tissues including platelet, brain, vas deferens, spleen, lung, heart, liver, kidney, blood vessels, pancreatic islets

and the gastrointestinal tract, az-ARs have been shown to mediate plateIet aggregation, neurotransmitter release, vasoconstriction, regulation of ion secretion and inhibition of insuhn secretion. As for the ai-AR, pharmacological and functional studies have also suggested heterogeneity of a2-AR. (for review see Ref. 55) Different rank orders of potency for adrenergic tigands at a2-adrenergic binding sites in various tissues have been described. The compounds prazosin, oxymetazoline, ARC 239 and chlorpromazine discriminate between two different subtypes termed a2A and a28 [56-60]. The selectivity of the drugs varies from a high c 100-fold for prazosin and ARC239, to a relative low ~ 18-fold for chlorpromazine. Competition radioligand binning experiments performed with these compounds have identified the human colonic carcinoma cell line HT29 and human platelet as containing only the a2A subtype, while the neuroblastoma× glioma hybrid cell line NG108-t5 and neonatal rat lung tissue contain only the a2B subtype [56]. Rat cerebral cortex appears to contain both subtypes [57]. The ratio of Ki values for prazosin and yohimbine (praz/yoh) has also been used as a criterion for classification into the aza or a2B subtypes. The praz/yoh ratio for the a ~ subtype is 240 (platelet), 570 (HT29), while that for the a2B is 5 (both neonatal lung and NG108-15) [61]. A third pharmacologically defined a2-adrenergie receptor subtype has been described in the opposum kidney derived OK cell line [61]. This receptor is similar to the azB subtype as it has relatively high affinity for prazosin, chlorpromazine and ARC 239, and tow affinity for oxymetazoline. However, the praz/yoh Ki ratio is 40; intermediate between that of the az~ (300-600) and otzB [5] subtypes. In addition, comparison of the pK i values of various drugs (including the aforementioned selective compounds) for the a2-adrenergic receptors in OK cells with those for the aza and a2B subtypes gives relatively poor correlations (approx. 0.8). Therefore, the a2-adrenergic receptor in OK ~ells has been designated as the azc subtype. Finally, a fourth subtype has been described in rat submaxillary gland which appears similar to the aa-adrenergie receptor found in the bo:-:ne pineal gland [62,63]. This receptor is characterized by having a higher affinity for phentolamine and lower affinity for rauwolscine and SKF104078 than the three other subtypes. The correlations between pK i values of various subtype selective drugs from the bovine pineal with those of the a ~ (HT29 0.80), the a2B (NGI08-15 0.46) and a2c (OK 0.61), have suggested that the bovine pineal and rat submaxillary gland a2-adrenergic receptor be designated as a fourth subtype: the a2D. The compound SKF 104078 has been reported to discriminate between the pre and postsynaptie a 2adrenergic receptors [64]. The a ~ , a2u and a2c subtypes all have high affinity for this compound suggest-

134 lag that they are all Oostsynaptic. Interestingly, the bovine a2D subtype has low affinity, suggesting that it may represent the presynaptic receptor. However, the utility of SKF 104078 as a discriminator for the pre and postsynaptic az-adrenergic receptor is unclear. At least one group has failed to confirm that this compound is setective for the postsynaptic receptor, and in fact their results suggest that both the a2A and a2B-adrenergic receptors are located presynaptically in some tissues [65-67]. Thus, on the basis of pharmacological profiles one would postulate four distinct a2-adrenergie receptor subtypes. The definitive classification of these receptors into pre or postsynaptic types awaits further investigation.

IliA. Elucidation of the primary structure of az-adrenergic receptors The first a2-AR gene to be isolated was that encoding for the human platelet receptor [5]. Somatic celI hybrid analysis revealed that this gene resided on human chromosome 10. Therefore, this receptor was given the designation azCl0. A human genomie Southern blot probed with a 0.95 kb fragment of the a2C10 exon revealed the presence of three related genes, each localizing to a different chromosome; 10, 4 and 2 [5]. Because of the relatively stringent conditions under which these experiments were performed these results did not prove but constituted highly suggestive molecular evidence for the existence of multiple a2-AR. This same 0.95 kb fragment of a2C10 was used to isolate, from a human kidney eDNA library, a distinct ot2-AR whose gene resides on chromosome 4, and is termed ot2C4 [6]. Recently, a third human ot2-AR gene located on chromosome 2 (azC2) has been isolated using an approach based on the polymerase chain reaction [7]. Oligonucleotide primers were synthesized corresponding to various conserved regions of a2Clt'l and a2CA. These primers were used to amplify, from human genomic DNA, a portion of a novel t~z-AR gene which was then employed to screen a human genomie library for a full length clone. A eDNA clone from rat kidney, designated pRNGa2, has also been recently isolated which has high homology, approx. 84% overall amino acid identity with a2C2 [8]. This done most likely represents the rat homologue of a2C2. The ligand binding characteristics of the three human az-adrenergic receptors expressed in COS-7 cells are shown in Table 1. As described for the a2A subtype, a2C10 has high affinity for oxymetazoline (K~ = 13 nM), low affinity for prazosin (K i ~ 2240 nM) and a K i ratio of prazosin/yohimbine of 450. a2C4 has a relatively intermediate affinity for oxymetazoline (Ki = 125 nM) and a high affinity for prazosin (K i = 68 nM) such as that described for the a2B and a,_c subtypes. The K~ prazosin/K~ yohimbine ratio of 14 is intermediate

between that described for a2a, 5 and a2c, 40. ot2C2 has the lowest affinity for oxymetazoline (K i ~ 1500 riM), a relatively high affinity for prazosin (K i = 293 riM) and a a~B-like K i prazosin/K i yohimbine ratio of 29. AII three of the cloned human receptors have relatively low affinity for phentolamine and high affinity for SKF 104078, suggesting that they do not represent the described a2o subtype. Corynanthine, WB410I and epinephrine distingt,ish a2C2 from rt2C4, prazosin and p-aminoelonidine distinguish a2C2 from a2C10, while corynanthine and prazosin discriminate between a2C4 and ~2C10, Oxymetazoline appears to be the single most useful drug as it discriminates between all three receptors, having approx, a 10-fold difference in affinity for each receptor.

III-B. Tissue distribution of the cloned a2-adreuergic receptors In order to better assign these receptors to the pharmacologically defined subtypes, we performed Northern blot analysis on tissues and cell lines which express each subtype (Table !1) [68]. A probe from the a2Cl0 receptor gene was able to hybridize at high stringency to mRNA from the human colonic carcinoma cell line HT29 and to rat cerebral cortex, two models for the a ~ subtype. Based on ligand binding properties and tissue localization studies, a2C10 Can confidently be assigned to the ¢d2Asubtype. Expression of a2C4 was detected in regions of rat brain and in the neuroblastoma × giioma NO 108-15 cell line, but not in neonatal rat lung: all tissues being models for the nab subtype. In addition, the azCA probe also hybridized to a mRNA species in the opossum kidney.derived cell line OK cell, a model for the a2c subtype. The pharmacologic profiles between the a2c and the a2B receptors differ only slightly. The azc affinity for yohimbine (K i ~ 0.23 nM) differs from that of azB (K i = 0.67 NG 108-15, 0.99 neonatal rat lung), and the a2c prazosin/yohimbine ratio [40] is intermediate between the az~ (570 HT29, 240 platelet) and the a2B 5.4. However, the pharmacologically defined a2c and a2B subtypes resemble each other in that the absolute affinities of the subtype selective drugs prazosin, ARC-239 and oxymetazoline, the rank orders of potency yohimbine > prazosin = phentolamine > ehlorpromazine = corynanthine, and the ratios of K i values for various subtype selective drugs are similar. Therefore, it appears that a2C4 may represent the described a2c subtype in OK cells and the a , B subtype found in the rat central nervous system, but not the a2B subtype found in peripheral tissues such as neonatal rat lung. Northern blots suggest that a2C4 also represents the a2B NGI08-15 cell receptor. However, pharmacological data demonstrate that this receptor is

135 most similar to the rat ne~,r,tal lung receptor, not the OK cell receptor. Therefore, definitive assignment of the NG108-I5 az-AR should await further study. a2C2 was found to be expressed in adult rat liver and lung but not in neonatal rat lung. This result was surprising since a2C2 has an a2B like pharmacology and lacks potential sites of N-linked glycosylation.The receptor in neonatal rat lung not only has the pharmacology of an azB receptor, but has also iaeen shown to be nonglycosylated[69]. In fact, the aforementioned rat receptor pRNGa 2, whic, has structural and ligand binding characteristics very similar to that of a2C2, has been shown to hybridize to mRNA from rat neonatal lung and adult rat kidney [8]. This would suggest that c¢2C2 does represent the a2a receptor in rat neonatal lung. With the isolation of three di,,.:,inet a2-adrenergic receptor genes, we are able to propose a new and more accurate classification (Table IV). The cloning of the genes has enabled the determination of the K i values for various subtype seIective drugs from radioligand binding experiments performed on membranes which contain only one of the three receptors, avoiding the possible complications of determining Ki values from tissues which may express multiple receptor subtypes. In addition, all three receptors have been isolated from the same species (human) and expressed in the same tissue (COS-7 cells). The receptor distribution has been determined in various tissues by performing Northern blot analysis. Finally, important structural information has been gleaned from the deduced amino acid sequences. The pharmacologic data coupled with the tissue localizations, and the structural information has led us to propose the following merger of the molecular and pharmacologic classifications: the azCl0 receptor is undoubtedly the a2A receptor (both HT29 and human platelet) as described in the pharmacologic literature. The a2C4 receptor is likely the aza receptor from rat cerebral cortex and possibly also from NG10815 cells, and the previously proposed a2c receptor from OK cells. In order to keep the classification

meaningful we propose to designate the a2C4 receptor as the azc subtype. Hopefully, this designation will clearup the confusion over this receptor, as it has been previously classified as an a2a, an azc, and a non a_,A, a~B or a~c receptor. Finally, the azC2 (and pRNGa 2) receptors represent the a2B receptor found in neonatal lung tissue. III-C. Gene organization and chromosomal localization

All thrcc of the a2-adrenergic receptor genes are intronless. As previously described each receptor subtype is localized to a different human chromosome: 2, 4 and 10. The only described promoter region for any of the a-adrenergic receptors is that for the human a2CI0 [70]. Approx. 2000 bp immediately 5' to the initiator methionine have been sequenced. Analysis of this region has revealed the presence of multiple potential regulatory elements including TATA boxes, a reverse complement of the CAAT consensus sequence, a SP1binding sequence and a steroid receptor-binding hexamer. Definitive assessment of whether these elements may actually regulate gene expression of a2-adrenergic receptors awaits further studies. III-D. Structure of a sadrenergic receptors

The deduced amino acid sequences of the three human a2-ARs range from 450 to 461 residues in length and are depicted in Fig. 2. The deduced amino acid sequences of a2C4 and azC10 as shown in Fig. 2 are slightly different than the deduced sequences as originally reported [5,6]. Nucleotide sequencing errors occurred in the GC rich regions of the genes encoding for the putative third intrao/toplasmic loops. The corrected regions of the sequence are clearly outlined in the legend to Fig. 2. The sequencing errors were identiffed by the cloning of the genes from other species [71-73], and the human genes/cDNAs were resequenced and confirmed in our laboratory (Ostrowski, J., Kurose, T., Lomasney, J.W., Caron, M.G. and

TABLE IV

Proposed az.adreoergic receptor classification Subtype Human chromosome AgonisTs Antagonists Tissue localization

Ot2A I0 O ~ ~, Epi > NE Rau :~) Praz cerebral cortex human platelet

¢tZB 2 Oxy = Epi _>NE Rau :~ Praz liver kidney

o'2c 4 Oxy = Epi > NE Rau : ) Fraz cerebral cortex OK

~t2n

HT29

neonatallung

NGI08-15?

Glycosylation sites SKF 1040"/8

yes high

of, h~,gh

yes high

? low

Aminoacids

450human(5, 70)

450human(7, 84)

461human(6)

?

450 porcine (73) 4.50 rat (85)

453 rat {8)

458 rat (71, 72)

Oxy ~> NE Rau > Praz rat submaxillary bovine pineal

136 Lefkowitz, R.J., unpublished results). The putative topographies are similar in that all three receptors have relatively long third intracytoplasmic loops and short carboxyl-termini. The MSD are strikingly conserved having 75% amino acid identities. Multiple serine and threoninc residues present in the third loops may be sites for phosphorytation by regulatory kinases such as //-adrenergic receptor kinase, which has been shown to phosphorylate a2C10 [74]. The amino terminus of otzC2 is among the shortest (12 amino acids) of the members of the G-protein coupled receptors and lacks sites for N-linked glycosylation which are present in both a~C4 and azCl0, a2C4 is the only adrenergie receptor which lacks the highly conserved cysteine residue in the carboxyl-terminus and therefore is presumably not palmitoylated.

IlI-E. Signal transduction of az-adrenergic receptors To date in virtually every system examined the activation of a:-AR inhibits adenyiyl cyclase. However, in many cases a2-AR-mediated physiological effects cannot be solely explained by a decrease of intracellular cAMP. The activation of several signalling pathways has been reported for the a2-AR, including the activation of K + channels [75], the inhibition of Ca2+ chan-

nels [76], increased Na+/H ÷ exchange [77] and mobilization of intracellular Ca2÷ [78]. Activation of a2C10, a2C4 and ot2C2 expressed in different fibroblast cell lines decreased both basal and forskolin-stimulated adenylyl cyclase activity via a PTX-sensitive G-protein (Ref. 51, Lomasney, LW., Caron, M.G. and Lcfkowitz, R.J., unpublished results). Interestingly, stimulation of a2C10 showed a 2-fold greater inhibition of basal adcnylyl ¢yclase activity than a2C4. This result may reflect differences in the molecular interactions of a2C10 and t~2C4 with the inhibitory G-proteins, though reconstitution studies with the purified a2C4 and a2C10 receptors and different recombinant ~i proteins have shown similar rank orders of coupling efficiency (Gi3 > Gil > Gi2) for each receptor [79]. In addition to inhibition of adenylyl cyclase, stimulation of both thC10 and azC4 results in a direct activation of PLC, via a PTX.sensitive G-protein [51]. The pattern of biochemical events produced by the ot2-AR is similar to that reported for the M2 and Ma muscarinie and for the 5HT1A receptors, which can both inhibit adenylyl cyclasc and activate PLC [80,81]. The cloning and expression of different receptor subtypes has provided the first direct evidence that a single receptor subtype can couple to multiple effcctor sysM.I

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269-300havebeencorrectedfor a:C4and residues332-364for a2C10(see text).

137 toms, suggesting a growing heterogeneity of receptoreffeetor interactions. Receptor chimeras have proven very useful in the identification of the various functional domains of Gprotein coupled receptors. This approach allows one to draw positive inferences about the function of specific receptor domains circumventing the problems associated with the interpretation of functional loss after deletional mutagenesis. In fact the very first receptor chimeras were constructed from the//2 and av,(Cl0) receptor genes [34]. These studies clearly implicated the fifth and sixth transmembrane spanning domains along with the third intracellular loop as the domains primarily responsible for conferring specificity of G a coupling. In addition, these studies demonstrated the importance of the seventh transmembrane spanning domain in determining agonist and antagonist speeifiery for the a 2 and 132-adrenergic receptors. Additional ~2-adrenergic receptor chimeras, which contained 12-22 amino acid substitutions with corresponding sequence from the N- and C-terminal portions of the third intraeellu!ar loop and proximal cytoplasmic tail of the a2Cl0 adrenergic receptor, have confirmed that these regions are involved in determining the specific G-protein: receptor coupling [82]. These studies also demonstrate the interaction of multiple cytoplasmic receptor domains for receptor:G-protein coupling. Subsequent work with other G-protein coupled receptors has shown that the functional domains of the a 2 and #2-adrenergic receptors can be generalized to other members of the G-protein coupled receptor supeffamily. Ligand binding to a2-adrenergic receptors has been shown to be modulated allosterically by monovalent cations [83]. Studies with the porcine brain azn adrenergic receptor has shown that the allosteric modulation still occurs with the purified and trypsinized hydrophobic core of the receptor [84]. Therefore, the site of modulation by monovalent cations occurs in the receptor, not in the associated G-protein subunits, and more specifically in the transmembrane spanning domains of the receptor. An aspartic acid residue located in the second transmembrane spanning domain of the a ~ adrenergic receptor appears to be the specific site of interaction with the monovalent cation Na + [86]. If this residue (Asp-79) is changed to an asparaginc, the resulting mutant az-adrenergie receptor, while binding ligands like the native receptor, is not modulated by Na +. It is likely that the carbox'yl-group of Asp-79 serves as a counter ion for the monovalcnt cations, thus serving as the site of interaction. Since this Asp residue is conserved among many members of the G-protein coupled receptor family, it is possible that this residue may mediate the modulation of ligand binding by monovalent cations at other G.protein coupled receptors.

IV. Conclusions The adrenergic receptors have served as excellem models for the study of receptors coupled to guanine nueleotide-binding regulatory proteins (G-proteins). The genes/cDNAs foc trine adtenergic receptors including three a t and three a 2 subty0cs have been isolated to date. This has allowed much more extensive characterization of the structure, pharmacology and tissue distribution of the receptors. The information gained has not only revised the present classifications, but has also provided hope that more selective and specific therapeutic agents may be developed. References | Birnbaumer. L., Abramowitz, ]. and Brown, A.M. (1990) Biochim. niophys. Acta 103l, 163-224.

2 Dixon, R.A.F., Kobilka, B.K., Strader, DJ. Banovic, J=L1 Dohlman, H.G., Frielle, T., Bolanowski, M.A., Bennett, C,D,, Rands, E,. Diem R,E., Mumford, R.A.. $1ater, E.E., SigaLLS., Caron, M.G., Le~owitz, ILL and StradeL C.D. (1986) Nature 321, 75-79. 3 Frieile, T., Collins, S,, Daniel, K_W., Carom M,G,, Lelkowit~ R.J. and Kobilka. B.IC (1987) Prec. Natl, Acad. Sci. USA 84, 7920-7924. 4 Emorine,L.J,, Marulto,S., Brieno-Sutren,M-M.,Patty, G., Tate, K., Delavier-Klutchko,C. and Strosbcrg.A.D.(1989)Science245, II18-1121.

5 Kobilka,B.K.,Matsui,H., Kobilka,T.S., Yang-Feng,T.L, Franke, O., Caron. M.G., 1.,¢fkowitz,RJ. and Regan,J.W. 0987) Science 238. 650-656 6 Regan. J.W., Kobilka.T.S., Yang-Feng,T.I, Caron, M.G. and L~fkowitz, RJ. and Kobilka. I~IC (|988) Prec. Natl. Acad. Sci. USA 85, 6301-6305. 7 Lomasney,J.W., Lorenz, W., Allen, LF., King,IC Regan, J.W., Yang-Feng, T.L., Cat'on, M.G. and Let'kowitz,RJ. (1990) Proc. Natl. Acad. Sci. USA 8% 5094-5098. 8 Zeng, D.. Harrison, J.K. D'Angelo.D.D,, Barber, CM., Tucker, A.L. Lu, Z. and Lynch,1CR.(1990) Prec. Natl. Acad. Sci.USA 87. 3102-3106, 9 Colecchia. S.. Schwinn, D.A., Randall. R,R., Lefl~owitz,RJ,, Caron, M.G. and Kobilka,B.K.(1988)Prec, Nail. Acad.Sci.USA 85, 7159-7163. 10 Schwinn, D.A.. Lomasney,J.W., Lorenz, W. Szklut, PJ., Fremeau, R.T., Jr., Yang-Feng,T.L., Cat,on, M.G,. LeAowitz,R.J. and Cotecchia,S. (1990) J. Biol.Chem. 265, 8183-8189. It Lomasney, J.W., Cotecchia, S., Lorenz, W,) Leung. W-Y,, Schwinn. D.A., Yang-Feng. T.L., Brownstein,M., Letkowitz,RJ. and Caron, M.G,(1991) L Biol.Chem.266,6365-6369. t2 Letkowitz, R,.I. and Caron, M.G. (Iqgg) J. Biol. Chem. 263. 4993-4996.

13 Dohlman, H.G., Camn. M.G. and Leftowitz, RJ. (1987) Biochemistry 26, 2o57--2664. t40'Dowd. B.F., Leikowitz, RJ. and Caron, M.G. 0989) Annu. Rev. Neurosci. 12, 67-83. 15 Findlay.J.B.C. and Pap#n, DJ.C. (1986) Biochem.J. 238, 625642, 16 Henderson, P. and Unwin,P.N. (1975) Nature 257, 28-32. 17 Henderson, P., Baldwin, J,M., Ceska, T.A., Zemtin, F.. Beckmann, E. and Downing.ICH,0990) J. Mnl. Biol.213, 899-929. 18 Hargrave, P.. Mcdowell,J.H., Feldman, RJ., Atkinson, P.H., Rap, J.K.M. and Argos, P. 0984) Vision Re,s. 24, 1487-1493.

138 t9 Dohlman, H.G., Bouvier, M.. Benovic, J.L., Caron, and Lelkowitz, R.J. (1987)J. Biol. Chem. 262, [4282-14288, 20 Wang, H-Y., Lipfert, L, Malbon, C.C. and Bahouth, S. (1989)J. Biol. Chem. 264. 14424-14430. 21 Dixeo. R.A.F., Sigal, 1.S. and Strader, C.D. (1988) Cold Spring HarLot Symposium Quant. BioL 53, 487-497. 22 Morrow, A.L. and Creese, 1. (1986) MoL Pharmaeol. 29, 321-330. 23 |lanfi, G., Gro~, G., Beckeringh, J.J. and Korstanje, C. (1989) J. Pharm. PharmacoL 41,714-716, 24 Gross. G.. Hanfl. G. and Ruggvics, C. (1988l Ear. J. Pharmacol. 151,333-335. 25 Boer, R., Grassegger, A., Schndl, C. and Glossmau, H. (1989) Ear. J. Pharmacol. 172, 13[-145. 26 Michel. A.D., Loary, D.N. and Whiting. R.L. (1989) Br. J. Pharmacol. 98, 883-889. 27 Hart. C., Abe[, P.W. and Minueman, K.P. (1987) Mol. PharmacoL 32. 505-510. 28 Han. C, Abel, P.W. and Miuneman, K,P, ([987) Nature 329, 333-335. 29 Minneman. K.P., Han, C. and Abel, P.W. (1988) Mol. Pharmacol. 33, 509-514. 30 Schwian, D.A., Page, S.O., Middleton, J.P. Lorenz, W., Liggett, S.B., Yamamoto, K., Caron, M.G., Leikowitz, R.J. and Cotecchla, S. (1991} Mol. PharmacoL, in press. 3! Yang-Feng, T.L., Xue, F.. Zhong, W., Cotecchia, S., Frielle, T., Caron, M.G., Letkowitz, g J . and Franke, U. (1990) Proc. Nail, Acad. Sc[. USA 87, 1516-1520. 32 K~e, J. and Do,alittle, R.F. (1982) J. Mol. Biol. 157, 105-132. 33 Strader, C.D., Dixon, R.A,F, Cheung, A,K., Candelore, M.R., Blake. A.D.. Sigal. I.S. (1987) J. Biol. Chem. 262, 16439-16443. 34 Kobilka. B.K.. Kobiika. T.S., Daniel. K, Regan, J.W., Caron, M.G. and l.,¢fkowitz, R.J. (1988) Science 240,1310-1316. 35 Dixon, R.A.F., SigaL I.S.. Rands. E.. Register. R.B.. Candetore, M.R, Blake, A.D. and Srader, C.D, (1987) Nature 326,73-77. 36 O, Dowd, B.F., Hnatowich, M., Regan, J.W., Leader, W.M., Caron, M.G. and Lelkowitz, RJ. (1988) J. Biol, Chem. 263, 15985- I5992, 37 Strader, C.D., Sigal, I.S., Register, R.B. Canelore, M.R,, Raads. E. and Dixon, R.A.F. (1987) Proc. Natl. Acad. Set. USA 84, 4384-4388. 38 Buckland. P.R, Hill, R.M.. Tidmarsh, S.F, and McGuffio, P. (1990) Nucleic Acid Rcs. 18, 682. 39 Leeb-Lundberg, L.M.F., Dickinson, K.E.J., Heald, S.L., Wikberg, J.E.S., Hagen, P-O., DeBernardis, J.E, Winn, M,, Arendsen. D.L, Lefkowitz, RJ. and Caron, M.O. [1984)J. Biol. Chem. 259, 2579-2587. 40 Lomasney, J.W.. Leeb-Lundberg, LM.F.. Cotecchia, S.. Regan, LW., DeBemardls. J.F., Caron, M.G, and l..¢fkowitz. R.J. (1986) J. Biol. Chem. 261, 7710-7716, 41 Sawutz, D.G,, Lanier, S.M., Warren, C.D. and Graham, R,M, (1987) Mol. Pharmacol. 32, 565-571. 42 Ovchinnikov, Y.A,, Abdulacv, N.G. and Bogachuk, A.$. (1988) FEBS Lctt. 230, 1-5. 43 O'Dowd, B.F., Hnalowitch, M., Caron, M.G., Lefkowitz, R.J. and Boavier, M. (1988) J, Biol. Chem. 264. 7564-7569. 44 L¢cb-Lundberg, L.M.F., Cotecchia, S., DeBlasi, A., Caron, M.G. and I.,¢fkowitz, RJ. 0987)J. Biol. Chem. 262, 3098-3105. 45 Bouvier, M., Leeb-Lundberg, LM.F., Benovic, J.L. Caron, M.G. and Lel'kowilz, RJ. (1987) J. Biol, Chum, 262, 3106-3113. 46 Leeb-Lundberg, L.M.F., Cotecchia, S., Lomasn~, J.W., DeBemardis, J.F., Lefkowitz, RJ. and Caron M.G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655. 47 Berridge, M.J. and Itvine, R.F. (1989) Nature 341, 197-205. 48 Minneman, K.P. (1988) Pharmacol, Res. ,40. 87-119. 49 Slivka, S.R., Meier, K.E. and Insel, P.A. 0988) J. Biol. Chem. 263. 12242-12246:. 50 Butch. R.M., Luini, A., Mats, D.E., Corda, D. Ve,nderhoek, J.Y.,

Kohn, L.D. and Axelmd, J. (1986) J. Biol. Chem. 261, 1123611241. 51 Cotecchia. S., Kobilka, B.K., Daniel, K.W., Nolan, R.D., Lapetina, E.Y., Carom M.G.. Le[kowitz, R.J. and Regan, J.W. (t990) J. Biol. Chem. 265, 63-69. 52 Cotecchia, S., Ostrowski, J., Kjelsberg, M., Caron, M.G. and Lefkowltz. RJ. (1991) J. Biol. Chem., in press. 53 Cotecchia, S., Exam, S., Caron, M.G. and Lefkowitz, R.J. (1990) Proc, Natl. Acad. Sci. 87, 2896-2900. 54 Voigt, M.M., Kispert, J., Chin H.M. (1990) Nucleic Acid Rcs. 18, 1053. 55 Docherty, J.R. (1989) Pharmac. Ther. 44, 241-284. 56 Bylund, D.B., Ray-Prenger, C. and Murphy, T.J. (1988) J. Pharmacol. Exp. Ther. 245, 600-607. 57 Bylund, D.B. (1985) Phannacol. Biochem. Behav. 22, 835-843. 58 Bylund, D.B. (1988) Trends Pharmacoi. Sci. 9, 356-361. 59 Cheuug, Y-D., Barnett, D.B. and Nahorski, S.R. (1982) Eur. J. Pharmacol. 84, 79-85. 60 Nahorski, S.R., Barnett, D.B. and Cheung, Y-D. (1985) Clio. Sci, 68 (Suppl. 10), 39s-42s. 61 Murphy, T.J. and Bylund, D.B. (1987) J. PharmaeoL Exp. Ther. 244, 571-578. 62 Michel, A.D., Loury, D.N. and Whiting, R.L. (1989) Br. J. Pharmaco]. 98, 890-897. 63 Bylund, D.B., Blaxall, H.S., Murphy, T.J. and Simmoneaux, V. (1991) in Adrenoceplors: Stucture, Mechanisms, Function (Szabadi, E., ed.), pp. 27-36, Birkhauser Verlag, Basel. 64 Hieble, J.P., Sulpizio, A.C., Nichols, AJ. Demarinis, R.M., P[eiffur, F.R., Lavanchy, P.G., and Ruffulo, R.R. (1986) ,L Hypcrtcnsion 4, (suppl. 6) 5189-5192. 65 Connaughton, S. and Docherty, J,R. (1988) Naunyn-Schmiedebore's Arch. Pharmacol. 338, 379-382. 66 Connaughton. S. and Docherty. J.R. (1990) Br. J. Pharmaeol. 99, 97-102, 67 Connaughtou, S. and Docherty, LR. 0990) Br. J. Pharmacol. lOl, 285-290. 68 Lorenz, W., Lomasney, J.W., Collins, S., Regan, J.W., Caron, M.G. and Le~owitz, RJ. (1990) Mol. PharmacoL 38, 599-603. 69 Lanier, S.M., Homey, C.J., Patenande, C. and Graham, R.M. (1988) J. Biol. Chem. 263, 14491-14496. 70 Fraser, C.M, Arakawa, S., McCombie, W.R. and Venter, J.C. (1989) ,I. Biol. Chem. 264, 11754-11761. 71 Flordellis, C.S., Hand,/, D.E., Bresnahan, M.R., Zannls, V.L and Gawas, H. 0990) Proc. Natl. Acad. Sci. USA 88, 1019-1023. 72 Voigt, M.M., McCuue, S.K., Kanterman, R.Y. and F¢lder, C.C, (1990) FEBS Lett. 278, 45-50. 73 Guyer, C.A., Horstman, D.A.. Wilson, A.L., Clark, J.D., Cmgoe, E.J. and Limbird, LE. (1990)J. Biol. Chem. 265, 17307-17317, 74 Benovic, J.L, Regan, J.W., Matsui, H., Mayor. F. Cotecchia, S., Leeb-Luudberg, L.M.F., Caron, M.G., and Lefkowitz, R.J. (1987) J. Biol. Chem. 262, 1725I- 17253. 75 Aghajanian G.K. and Vandermaelen, C.P. (1982) Science 215, 1394-1396. 76 Holz IV, G.G., Rane, S.G. and Dunlap, IC (1986) Nature 319, 670-672. 77 Isom, LL., Cragoe, E,J, and Limbird, LE. (1987) J, Biol. Chem. 262. 6750-6757. 78 Michel, M.C., Brass, L.F., Williams, A., Bokoch, G.M., LaMorte, V.J. and Mutalsky, HJ, (1989) J. Biol. Chem. 264, 4986-4991. 79 Kurose, H., Regan, J.W.. Caron, M.G. and Letkowitz, R.J. (1991) Biochemistry 30, 3335-3341. 80 Peralta, E.G., Ashkenazi, A, Winslow, J.W., Ramachandran, J. and Capon, DJ. (1988) Nature 334, 434-437. 81 Fargin, A,, Raymond, J.R., Regan, J.W. Cotecchia, S., Leikowitz, R,J. and Caron, M.G. (1989)J. Biol. Chem. 264, 14848-14852. 82 Liggett, S.B., Caron, M.G., Lefkowitz, R.J, and Hnatowlch, M. (1991) J. Biol. Chem. 266, 4816-4821.

139 83 Nunnari, J,M., Repaske. M.G., Brandon. S.. Cragoe. Jr.. Ed. and Limbird, L.E, (1987)J. Biol. Chem. 262, 12387-12392. 84 Wilson, A.L., Guycr. C.A., Crago, Jr.. EJ. and Limbird. L.E. (1990) J. Biol. Chara. 265, 17318-17322. 84 Weinshank, R.L., Zgombick..I.M.. Macchi. M.. Adham. N., Lichtblau, H., Branchek, T.A. and Harlig, P.R. (1990) Mol. Pharmacol. 38, 681-688.

85 Chalberg, S.C.. Duda, T.. Rhine, J.A. and Shanna R,K. (1990) Mol. Cellular Biochem. 97, 161-172. 86 Horstman, D.A., Brandon, S.. Wilson. A.L., Guyer, C.A., Cragoe. Jr.. E.J. and, Limbird, L.E. (1990).[. Biol. Chem. 265. 215(~)-21505.