insulinlike growth factor II receptors.

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Annu. Rev. Biochem. 1992.61:307-330. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 12/03/14. For personal use only.

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STRUCTURE AND FUNCTION OF THE MANNOSE 6-PHOSPHATE/INSULINLIKE GROWTH FACTOR II RECEPTORS Stuart Kornfeld Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 KEY WORDS;

mannose 6-phosphate receptors, insulinlike growth factor II receptor, Iysosomes, lysosomal enzyme

CONTENTS PERSPECTIVES AND SUMMARY ... .... . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . . . . ....

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INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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RECEPTOR STRUCTURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prima ry Structure and Genomic Organization....... . . . . . . . .. . . . . . . . . . . . . . . . . . . ... . . . . . . ... Oligomeric Structure .. . . . . . . . . ............................. . . . . . . .. ...... . . . . . .. . .. ..... . . . . . .... Ligand-B inding P ropertie s . . . .. . ......... . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .

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RECEPTOR FUNCTION .......... . . . . . . . . . . . . . . . . . . . . . . .. . . . Role in Lysoso ma l Enzyme Sorting and Endocytosis . . . . .. . . . . . . . . .. . .. ............. ...... Role in Signal Transduction ... . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . .... . .... ... . . . . . . . ... .. . . . . Role in Hormone Clearance and Activa tion. . . . . . . . .... . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role in Extracellula r Matrix Degradatio n ... . . . . . . . . . . . . . . . .................................

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RECEPTOR DISTRIBUTION AND TRAFFICKING . . . . . . .. . .. . ...... . . . . . . . . . . . . .. . . . . . . . . . Subcellular Localizatio n....... ......... . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. ............ . . . . . ..... Regulation of Recep tor Tra fficking . ... .. ................ S tructura l Determinants of Recep tor Tra ffickin g . . . . . . . . . . . . . . . . . . . ............ . .. . . . ...... Reconstitution of Receptor Tra fficking in Ce ll Extra cts . . . . . . . . . . . ..... . . . . . . . . . . ... . . . . . . . . . . . . . . . . .

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DEVELOPMENTAL REGULATION AND TISSUE-SPECIFIC EXPRESSION . . . . . . . . .

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PERSPECTIVES AND SUMMARY The discovery in 1987 that the cation-independent mannose 6-phosphate receptor and the insulinlike growth factor II receptor are the same protein raised the interesting possibility that this receptor functions in two distinct biologic processes, i.e. protein trafficking and transmembrane s ignal transduction. Cell transfection experiments have very recently provided direct evidence that the Man-6-P/IGF-II receptor mediates the transport of newly synthesized acid hydrolases to lysosomes. This function is shared with a second mannose 6-phosphate receptor that does not bind IGF-II. The eloning of cDNAs for these receptors has provided insights into their structures and has revealed that they are related proteins. The determinants on the receptors that mediate Man-6-P binding and direct their intracellular routing are being defined. A growing body of evidence indicates that the Man-6-P/IGF-ll receptor also functions in transmembrane signal transduction. However, this property appears to be confined to certain species and cell types. This receptor may have additional roles in the clearance and activation of other growth factors. This review focuses on recent findings concerning the structure, function, and cellular distribution of the Man-6-P/IGF-II receptors. This subject was last reviewed in 1989 ( 1 , 2). The reader is referred to these reviews as well as to several other recent reviews (3-7) for a more complete summary of the earlier work in this area.

INTRODUCTION Lysosomes are acidic cytoplasmic vacuoles that contain many hydrolytic enzymes that function in the degradation of internalized and endogenous macromolecules. Although the acid hydrolases themselves are relatively long lived proteins, they do tum over and must be replaced. Additionally , dividing cells must be able to synthesize new lysosomes. To meet these needs, higher cukaryotcs have developed an claborate system for targeting newly syn thesized acid hydrolases to lysosomes. With a few exceptions , the acid hydrolases are soluble glycoproteins that are synthesized in the rough endoplasmic reticulum where they undergo cotranslational glycosylation of selected asparagine residues. These early steps in the biosynthetic pathway are shared with secretory glycoproteins, and the two classes of proteins are transported together in vesicular carriers to the Golgi where they are sorted from one another for delivery to their final destinations. This sorting process is accomplished by the phosphomannosyJ recognition system. Through the concerted action of two enzymes , the acid hydrolases selectively acquire phosphomannosyl residues , which serve as high-affinity ligands for binding


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to the Man-6-P receptors (MPRs) in the Goigi. This step results in the physical separation of the acid hydrolases from the proteins destined for secretion. The ligand-receptor complexes are then gathered into clathrin coated pits and bud from the Golgi in coated vesicles that transport the acid hydrolase-receptor complexes to an acidified endosomal (prelysosomal) com partment. The low pH of this compartment induces dissociation of the ligand, which is then packaged into a lysosome. The receptor either returns to the Golgi to repeat this process or moves to the plasma membrane where it functions to internalize exogenous lysosomal enzymes and IGF-II or, in some instances, to mediate a transmembrane signaling event upon the binding of IGF-II.

RECEPTOR STRUCTURE Primary Structure and Genomic Organization Two distinct MPRs have been isolated, characterized, and their cDNAs cloned. One is a type I integral membrane glycoprotein with a Mr of 275,000. This receptor binds Man-6-P-containing ligands independent of divalent ca tions, and therefore has been called the cation-independent (CI)MPR. Howev er, since this receptor also binds IGF-II, it will be referred to as the Man-6-PI IGF-II receptor. The other receptor is also a type I integral membrane glycoprotein with a subunit Mr of approximately 46,000. The bovine and murine forms of this receptor require divalent cations for optimal ligand binding, so this receptor is referred to as the cation-dependent (CD)MPR (8-11 ). However, the human and porcine forms of the receptor have little or no requirement for divalent cations (12-14). cDNAs for the Man-6-P/IGF-II receptor have been cloned from bovine (15, 16), human ( l7, 18), and rat (19) sources, while cDNAs for the CD-MPR have been cloned from bovine (20), human (21), and murine (11, 22) sources. The amino acid sequences deduced from these cDNAs have provided insights into the structures of the receptors and have revealed that the two receptors are related proteins. The bovine Man-6-P/IGF-II receptor consists of four structural domains: a 44-residue amino-terminal signal sequence, a 2269-residue extracytoplasmic domain, a single 23-residue transmembrane region, and a 163-residue car boxyl-terminal cytoplasmic domain. The extracytoplasmic domain contains 1 9 potential Asn-linked glycosylation sites, a number of which are utilized , yielding a mature receptor of 275-300 kDa. Glycosylation is not required for the receptor to bind IGF-II (23). The extracytoplasmic domain also has a repetitive structure consisting of 15 contiguous repeating segments of approx imately 147 amino acids each. Each repeating segment shares sequence identities with all the other repeats, with the percent of identical residues ranging from 16 to 38%. In addition, there are many conservative amino acid


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substitutions. The location of the cysteine residues is highly conserved among the repeating segments. One unusual feature is that the 1 3th repeat from the amino terminus contains a 43-residue insertion that is similar to sequences found in fibronectin, factor XII, and a bovine seminal fluid protein (24). This segment forms part of a collagen-binding domain in fibronectin, but its function in the Man-6-P/IGF-II receptor is unknown. This is the only se quence in the receptor that is similar to sequences in other known proteins. The cytoplasmic domain of the receptor contains four regions with se quences that are known to be potential substrates for various protein kinases , including protein kinase C, cAMP-dependent protein kinase, and casein kinases I and II (19). These sequences are highly conserved among the bovine, human, and rat receptors, whereas the intervening sequences are quite degenerate (91% identity versus 38% for the intervening sequences). The receptor is known to be phosphorylated at a number of these sites (25-27). Overall, the amino acid sequences of the bovine and the human receptors are 80% identical. The Man-6-P/IGF-II receptor from embryonic bovine tracheal cells and embryonic human skin fibroblasts has been shown to contain covalently bound palmitic acid, most likely in an amide-linkage (28). The functional significance of this modification is unknown. The bovine CD-MPR also consists of four structural domains: a 28-residue amino-terminal signal sequence, a 159-residue extracytoplasmic domain, a single 25-residue transmembrane region, and a 67-residue carboxyl-terminal cytoplasmic domain. This receptor contains five potential Asn-linked glycosylation sites, four of which are used (20, 29). Alignment of the bovine, human, and mouse sequences reveals that the mature proteins are 93-95% identical, with the cytoplasmic domains being 100% identical. The cytoplas mic domain contains a single casein kinase II site. When these sequences are compared to the sequences of the Man-6-P/IGF-II receptor, it is evident that the entire extracytoplasmic domain of the CD-MPR is similar to each of the repeating units of the Man-6-P/IGF-II receptor, with sequence identities ranging from 14 to 28%. This suggests that the two receptors may be derived from a common ancestor, with the Man-6-P/IGF-II receptor arising from multiple duplications of a single ancestral gene. In contrast to this homology, there are no sequence similarities among the signal sequences, transmem brane regions, and the cytoplasmic domains of the two receptors. The gene for the human CD-MPR has been mapped to chromosome 12 (21), and the genomic structure has been determined to consist of seven exons (110-1573 basepairs) spanning more than 12 kilobases (30). Exon 1 contains 5' untranslated sequence, exon 2 encodes the signal sequence and the begin ning of the luminal domain , and exons 3-5 encode the remainder of the luminal domain. Exons 5 and 6 encode the transmembrane domain, and exons



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6 and 7 encode the cytoplasmic domain. Interestingly, the cysteines thought to be involved in disulfide pairs are located on different exons. This indicates that the intron/exon borders of the CD-MPR gene do not reflect the protein domains. The genomic structure of the Man-6-P/IGF-II receptor has not been elucidated as yet, but the gene has been localized to the long arm of human chromosome 6, region 6q25---,>q27, and mouse chromosome 17 , region A-C (31 ). Thus the two genes for the human Man-6-P receptors are on different chromosomes.

Oligomeric Structure The oligomeric structure of the CD-MPR has been analyzed in several laboratories ( l 0, 32-36). These reports indicate that detergent-solubilized receptor can exist as a monomer, dimer, or tetramer depending on the experimental conditions. The formation of dimeric and tetrameric forms is favored by low temperature «16°C), neutral pH, the presence of Man-6-P, and high protein concentration, whereas monomer formation is favored by higher temperatures, low pH, and low receptor concentration (35). The kinetics of dissociation and reassociation are relatively rapid at 37°C , raising the possibility that the receptor may undergo these transitions as it cycles to different cellular compartments. In the studies that utilized the human recep tor, the monomeric form did not bind to a phosphomannan-Sepharose affinity column (35). However, the monomeric form of the bovine receptor did bind to a pentamannosephosphate-affinity column when Mn2+ was present 00). Further evidence that the monomeric form of the bovine receptor can fold into an independent ligand-binding unit has come from studies of a truncated form of the receptor that lacks the transmembrane and cytoplasmic domains. This receptor, which behaves as a soluble monomer in solution, is capable of binding to a pentamannosephosphate-affinity column (33). Expression of a truncated form of the human CD-MPR gave rise to a soluble dimer under the conditions tested (36). These discrepancies may reflect species variation or possibly differences in the affinity column used to assess receptor-binding ability. The relevance of these findings to the state of the receptor in cellular membranes has yet to be established. Several studies have shown that the CD-MPR exists primarily as a dimer in the membrane as analyzed by chemi cal cross-linking agents (10, 32, 33). However, Waheed et al (35) observed monomeric, dimeric, and tetrameric forms of the receptor in baby hamster kidney cells overexpressing the CD-MPR. These authors speculate that changes in the quaternary structure of the receptor during recycling may influence the biologic behavior of this molecule. The quaternary structure of the Man-6-P/IGF-II receptor has not been

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analyzed as extensively, but hydrodynamic measurements are consistent with it being a monomer (37), while chemical cross-linking experiments indicate that it may be an oligomer (38).


Ligand-Binding Properties Equilibrium dialysis experiments indicate that the CD-MPR binds one mole of the monovalent ligand Man-6-P and 0. 5 mole of a diphosphorylated high mannose oligosaccharide per monomeric subunit (39 , 39a). Consequently each dimer would have two Man-6-P-binding sites , both of which can be occupied by a single oligosaccharide containing two Man-6-P residues. The Man-6-P/IGF-II receptor, on the other hand, binds two moles of Man-6-P or one mole of a diphosphorylated oligosaccharide per monomer (39a, 40). This suggests that only two of the 15 repeating segments of the receptor may function in the binding of Man-6-P. In this regard, two proteolytic fragments of the receptor corresponding to repeating units 1-3 and 7-11 have been shown to bind to Man-6-P-containing ligands (41). Therefore, these two regions of the receptor are likely to contain the two functional Man-6-P binding domains. Little is known about the actual binding sites for Man-6-P on either receptor. Wendland et al (42) mutated each of the five histidine and the eight arginine residues of the mature CD-MPR and found that His131 and Arg137 are essential for ligand binding. These investigators demonstrated that non glycosylated CD-MPR is stable and binds ligands with high affinity, whereas mutants with replacement of any of the six luminal cysteine residues with glycine residues are completely inactive (29, 43). This latter finding is consistent with the observation that newly synthesized CD-MPR polypeptides lack ligand binding and only acquire this property after formation of in tramolecular disulfide bonds (44, 45). While both receptors bind Man-6-P with essentially the same affinity (7-8 x 1 0 - 6 M), the Man-6-P/IGF-II receptor binds a diphosphorylated oligosac charide with a substantially higher affinity than does the CD-MPR (2 x 10 - 9 M vs 2 X 10-7 M , respectively) (39 , 39a, 40). Since oligosaccharides with two phosphomonoesters bind to the MPRs with an affinity similar to that observed for lysosomal enzymes, the high-affinity binding of lysosomal enzymes can be explained by a two-site model in which two phosphoman nosyl residues on the lysosomal enzyme interact with the receptor (40). This divalent interaction could either be mediated by two phosphomannosyl re sidues on the same oligosaccharide of the lysosomal enzyme or by phospho mannosyl residues located on different oligosaccharides . The latter interaction could result in an even better fit, resulting in higher-affinity binding. The secretion of the lysosomal enzyme cathepsin L by transformed mouse cells is



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of particular relevance in this regard. Dong et al (46) reported that NIH 3T3 cells transformed with the Kirsten sarcoma virus synthesize 25-fold more cathepsin L than nontransformed cells and secrete 94% of this enzyme while retaining most of the other lysosomal enzymes. The secreted cathepsin L, which contains a single oligosaccharide with two phosphomonoester moiet ies, bound to a MPR-affinity column with a l O-fold lower affinity than that observed with other lysosomal enzymes. Furthermore, cathepsin L syn thesized by Chinese hamster ovary cells bound to the receptor with higher affinity than mouse cathepsin L, and upon analysis was found to contain two oligosaccharides rather than the single one present on the mouse enzyme (46), with each oligosaccharide having two phosphomannosyl residues (47). These data are consistent with the view that individual phosphomannosyl residues located on different oligosaccharides interact with thc receptor with higher affinity than do two phosphomannosyl residues present on the same oligosac charide. On the other hand, if the membrane form of the Man-6-P/IGF-II

receptor proves to be a dimer (or the CD-MPR, a tetramer), then a four-site model for high-affinity ligand binding would be indicated and provide an explanation for the observed findings with cathepsin L. An alternative ex planation for the poor binding of mouse cathepsin L to the Man-6-P/IGF-II receptor has been put forward by Lazzarino & Gabel (48). These investigators found that intact procathepsin L, either native or in a reduced and alkylated state, has a poor affinity for the receptor, whereas phosphorylated oligosac chari des released from the enzyme bind to the receptor with high affinity. These results indicate that the polypeptide portion of cathepsin L contains determinants that inhibit binding of the phosphorylated oligosaccharide to the MPR. One possibility is that the negatively charged phosphomannosyl groups of the oligosaccharide interact ionically with positively charged lysine or arginine side chains of the protein backbone, and thereby are impaired in their interaction with the MPR. The bovine, human, and rat Man-6-P/IGF-II receptors all bind IGF-II, a nonglycosylated polypeptide, with high affinity (19, 49-53). In contrast, the chicken and frog receptors lack the high-affinity IGF-II-binding site (54, 55) as does the CD-MPR (49, 52). The stoichiometry of IGF-II binding to the bovine receptor has been determined to be one mole of ligand bound per polypeptide (49). Several studies have shown that the binding sites for Man-6-P and IGF-II on the receptor are distinct, and that the receptor can bind both ligands simultaneously (19, 49-52). However, lysosomal enzymes, in contrast to Man-6-P, do impair IGF-II binding, and IGF-II can inhibit lyso somal enzyme binding (19, 52, 56, 57). The significance of these inhibitory effects is unclear, since overexpression of IGF-ll in NIH 3T3 cells does not impair the sorting of newly synthesized lysosomal enzymes or the uptake of exogenous arylsulfatase A (58).

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RECEPTOR FUNCTION


Role in Lysosomal Enzyme Sorting and Endocytosis There is considerable evidence that the Man-6-P/IGF-II receptor functions in the sorting of newly synthesized lysosomal enzymes and in the endocytosis of extracellular lysosomal enzymes, whereas the CD-MPR only participates in lysosomal enzyme sorting under physiologic conditions. Thus, cultured cells that either lack endogenous Man-6-P/IGF-II receptor (59) or are depleted of this receptor with specific antibodies (4, 60) secrete 60-70% of their newly synthesized lysosomal enzymes and do not endocytose extracellular lysosom al enzymes. Transfection of the receptor-deficient cell lines with Man-6-PI IGF-II receptor cDNA results in receptor expression and correction of the defects in lysosomal enzyme sorting and uptake (61, 62). The residual sorting found in the Man-6-P/IGF-II receptor-deficient cells appears to be mediated by the CD-MPR, since treatment of the cells with anti-CD-MPR antiserum results in increased secretion of the acid hydrolases (60). When such cells are transfected with CD-MPR cDNA to achieve 5-20 times the level of endogenous receptor expression, the sorting of lysosomal enzymes increases to about 50% (11, 14). However, endocytosis of exogenous J3-glucuronidase is only observed in cells expressing 40-50 times the level of the endogenous CD-MPR, and even at these high levels of expression the efficiency of ligand uptake is only 1-2% of that mediated by physiologic levels of the Man-6-PI IGF-II receptor. The inability of the CD-MPR to function efficiently in endocytosis reflects its poor binding of ligand at the surface cell rather than its failure to recycle to the plasma membrane (11, 38, 60, 63). When the CD-MPR is overexpressed in baby hamster kidney (BHK) and mouse L cells that contain normal levels of endogenous Man-6-P/IGF-Il receptor, the sorting of lysosomal enzymes decreases from 90-95% to 50% (64). This effect requires the expression of high levels of CD-MPR that have the ability to bind ligand and to recycle. Cotransfection with Man-6-P/IGF-II receptor cDNA restores lysosomal enzyme sorting to the normal level. Chao et al (64) have proposed that the two receptors compete for lysosomal enzyme binding in the Golgi, with the ligands that bind to the CD-MPR being delivered to a site (early endosome or plasma membrane) from which they can be released from the cell. This interpretation would explain the inefficient sorting of lysosomal enzymes by Man-6-P/IGF-II receptor-deficient cells transfected with the CD-MPR eDNA.

Role in Signal Transduction The discovery by Morgan et al (17) that the CI-MPR and the IGF-II receptor are the same protein immediately raised the fascinating possibility that this receptor may function in two diverse biologic processes, i.e. protein traffick-



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ing and transmembrane signaling. As discussed in the preceding section, the evidence for a role in protein trafficking is well established. However, it has been more difficult to establish a role in signal transduction because IGF-II also binds to the IGF-I receptor and the insulin receptor, both members of the tyrosine kinase family of receptors known to transmit signals across the plasma membrane (5, 65-67). In fact, it has been shown that some effects of IGF-II on various cell types are likely to be mediated by its binding to the IGF-I receptor or the insulin receptor or possibly yet another receptor (6871). A clear example of IGF-II acting through a receptor other than the Man-6-P/IGF-II receptor is seen with chicken embryo fibroblasts. This cell type responds to IGF-II with enhanced protein synthesis and stimulation of cell division (72), even though the chicken CI-MPR does not bind IGF-II (54, 55). Nevertheless, a number of reports indicate that IGF-II does mediate responses through its own receptor. These responses include the stimulation of glycogen synthesis in rat hepatoma cells (73), the stimulation of amino acid uptake in human myoblasts (74), the promotion of cell proliferation in K562 cells (75), the stimulation of Na+ /H+ exchange and inositol trisphosphate production in canine kidney proximal tubular cells (76-79), the growth and development of rat metanephroi (80), and the stimulation of Ca2+ influx and DNA synthesis in BALB/c 3T3 cells (81, 82). In two of these studies it was shown that antibodies to the Man-6-P/IGF-II receptor mimic the action of IGF-II (73, 81), while in another study Man-6-P potentiated the stimulatory effect of IGF-II (78). In the study utilizing K562 erythroleukemia cells , it was shown that the target cells lack the IGF-I receptor, and their growth response to insulin and IGF-I was extremely poor relative to their response to IGF-II (75). Nishimoto and his colleagues have begun to elucidate the mechanism by which the Man-6-P/IGF-II receptor may transduce a signal across the plasma membrane. These workers found that IGF-II does not stimulate the opening of calcium channels or DNA synthesis in quiescent BALB/c 3T3 cells, but if the cells are first made competent by pretreatment with platelet-derived growth factor (PDGF) and then primed by a brief incubation with epidermal growth factor (EGF), IGF-II induces a twofold , sustained increase in calcium influx rate as well as an increase in [3H]thymidine incorporation into DNA (82). These biologic responses could also be induced by anti-Man-6-P/IGF-II receptor antibodies (81). The stimulatory effects of IGF-II on calcium influx were completely abolished by pretreatment of the cells with pertussis toxin, suggesting that the IGF-II effects were mediated by a mechanism involving coupling to a G protein. Evidence that this is the case was obtained by demonstrating that IGF-II brings about the direct coupling of Gi-2"" a Gi protein with a 40-kDa a subunit, with purified Man-6-P/IGF-II receptor reconstituted in phospholipid vesicles (83, 84). Gi-20' is a member of the


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membrane-associated oligomeric G proteins, which transduce receptor mediated signals to intracellular effectors (85). Interestingly, Man-6-P and Man-6-P-containing f3-glucuronidase did not stimulate GTPyS binding to Gj or activate its GTPase, but these agents completely inhibited the IGF-II induced Gj protein activation (86). These results indicate that the Man-6-PI IGF-II receptor has two distinct signaling functions that positively or nega tively regulate the activity of Gi-Za in response to the binding of IGF-II or Man-6-P. When a synthetic peptide corresponding to residues 122-135 of the cytoplasmic tail of the human Man-6-P/IGF-II receptor was incubated with Gi-zc" it activated the G protein in a manner similar to that observed with the intact receptor plus IGF-II (87). This region of the cytoplasmic tail was chosen for study since it shares the structural characteristics of mastoparan, a small peptide from wasp venom that mimics G-coupled receptors by directly activating G proteins (88). Peptides of similar size corresponding to other regions of the cytoplasmic tail had no biologic effects on these assays. The results are consistent with residues 122-135 of the cytoplasmic tail having a role in the Gi_2",-activating function of the receptor. The coupling of the receptor to Gi-Zct appears to be a regulatory step in the signal transduction process. Okamoto et al (89) have found that the failure of IGF-II to trigger the signaling pathway in quiescent BALB/c 3T3 cells is due to uncoupling of the receptor and Gj-2a. This coupling could be restored by incubating the cells with the combination of PDGF and EGF or by transfection with Kirsten sarcoma virus bearing the v-Ki-ras gene. However, it is not known whether ras p21 directly or indirectly acts on receptor-Gi_Za coupling. Okamoto et al (89) have proposed that Man-6-P/IGF-II receptor-Gi_Za uncoupling may be one mechanism that precludes quiescent cells from responding to IGF-II. S akano et al (89a) have taken a different approach to address the question of whether the biologic effects of IGF-II are mediated via the Man-6-P/IGF-II receptor or the IGF-I receptor. These investigators prepared IGF-I1 mutants that bind specifically to one or the other receptor. Thus, their Class I mutants with Tyr27 ....,. Leu or Val43 ....,. Leu substitutions bind with normal high affinity to the Man-6-P/IGF-II receptor and with extremely low affinity to the IGF-I receptor. In contrast, the Class II mutants with Phe48 , Arg49 , and Ser50 substituted with Thr, Ser and lie residues, respectively, or Ala54Leu55 substituted with Arg residues, bind to the IGF-I receptor with high affinity but very poorly to the Man-6-P/IGF-II receptor. These results show that the two receptors interact with different domains of IGF-II. Similar findings have been reported by others (89b, 89c, 89d). When the IGF-II mutants of Sakano et al (89a) were tested for their activity in two biologic systems (stimulation of DNA synthesis in BALB/c 3T3 cells and glycogen synthesis in Hep G2 cells), the induction of a biologic response correlated with high-affinity binding to

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the IGF-I receptor and not with binding to the Man-6-P/IGF-Il receptor. These results differ from the previous reports indicating that the biologic responses of these cells to IGF-Il is mediated by the Man-6-P/IGF-I1 receptor (73, 81). The Class I and Class II mutants of IGF-II appear to be promising reagents for analyzing the biologically important targets of this hormone.

Role in Hormone Clearance and Activation The Man-6-P/IGF-II receptor is known to bind and internalize IGF-II at the cell surface, resulting in the lysosomal degradation of this ligand (52, 90). In this manner the receptor may serve to clear IGF-II from the circulation. B inding of other hormones to the Man-6-P/IGF-II receptor at the cell surface may result in their activation. This appears to be the case with transforming growth factor-/31 (TGF-,Bl) precursor, the proform of a hormone that has multiple effects on cell growth and differentiation. This molecule has been found to contain Man-6-P residues and to bind to the Man-6-P/IGF-II receptor (91, 92). Dennis & Rifkin (93) reported that Man-6-P and anti-Man-6-P/IGF II receptor antibodies inhibit the activation of TGF-,BI precursor by bovine aortic endothelial cell/bovine smooth muscle cell co-cultures , suggesting that binding to the Man-6-P/IGF-1I receptor is required for latent TGF-,BI activa tion. It is not known whether this activation occurs at the cell surface or following internalization into acidified endosomes. Another growth factor that acquires Man-6-P residues is proliferin, a prolactin-related protein post ulated to be an autocrine growth factor (94). Its binding to the Man-6-P/IGF-1I receptor could result in its activation in endosomes or its degradation in lysosomes. Porcine thyroglobulin is a Man-6-P-containing glycoprotein that is secreted by thyroid follicle cells and then recaptured for degradation in lysosomes (95). The Man-6-P/IGF-II receptor may participate in its recapture and degradation.

Role in Extracellular Matrix Degradation Wang and colleagues have presented evidence that MPRs located at the cell surface contain bound acid hydrolases that degrade cell surface and sub stratum-attached proteoglycans (96 97). These investigators found that the addition of Man-6-P to human fibroblast cultures inhibited the turnover of extracellular 35S-labeled proteoglycans. When the experiment was performed with fibroblasts from patients with I-cell disease, which lack acid hydrolases carrying the Man-6-P marker, no difference was seen in the turnover of the proteoglycans with or without added Man-6-P. However, the addition of acid hydrolases derived from normal human fibroblasts to 35S-labeled I cells resulted in enhanced turnover of the proteoglycans, and this effect was inhibited by Man-6-P. These data suggest that the Man-6-P/IGF-1I receptor ,

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molecules at the cell surface serve to anchor acid hydrolases and thereby allow them to degrade pericellular and extracellular proteoglycans more effectively. If the acid hydrolases were acting in solution rather than while anchored at the cell surface, then the addition of Man-6-P, which releases the hydrolases from the surface receptors, should have enhanced rather than impaired the degradation of the proteoglycans.

RECEPTOR DISTRIBUTION AND TRAFFICKING Subcellular Localization The earlier studies on the distribution and trafficking of the MPRs have been reviewed (1), so this information will be briefly summarized and the focus will be on the new contributions in this area. There is general agreement that a single pool of MPRs cycle constitutively among the Golgi, endosomes, and the plasma membrane. Most of the biochemical evidence suggests that the major site for lysosomal enzyme sorting is the last Golgi compartment, variously referred to as the trans Golgi network (TGN), the trans Golgi reticulum, the trans tubular network, and GERL (Golgi endoplasmic reticu lum, lysosome) (98). In some cell types, such as pancreatic, hepatic, and epididymal cells, the Man-6-P/IGF-1I receptor has been found by im munolocalization techniques to be most concentrated in the early (cis) Golgi, raising the possibility that lysosomal enzymes may bind to the receptor in that compartment and either pass through the Golgi as a complex or exit the Golgi at the cis side of the stack (99, 100). In most cell types, this receptor is found primarily in the TGN, with lesser amounts detected throughout the Golgi stack ( 100-102, 102a). The immunolocalization studies have also revealed that at steady-state, most of the Man-6-P/IGF-I1 receptor is present in one or more populations of endosomes with very low, or undetectable amounts in structures identified as lysosomes (101-106). In normal rat kidney (NRK) cells, it has been estimated that 90% of the receptor is located in a late endosomallprelysosomal compart ment, with the rest being distributed over the plasma membrane , early endosomes, and the trans Golgi network (107). The intracelIular distribution of the CD-MPR has been examined in this same cell type ( 1 08). At steady state, this receptor is concentrated in the Golgi complex, mainly in middle and trans cisternae , whereas in cells treated with weak bases, the receptor redis tributes to endosomes that also contain the Man-6-P/IGF-ll receptor. The CD-MPR is also concentrated in the Golgi in Clone 9 hepatocytes and in exocrine pancreatic cells, whereas it is localized primarily in endosomes in U937 monocytes, mouse macrophages , and proximal tubule cells (108 , 109). In Madin-Darby Canine Kidney (MDCK) cells, which are polarized epithelial cells, the surface distribution of the Man-6-P/IGF-I1 receptor is exclusively


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basolateral (110). The CD-MPR could not be detected on the surface of this cell type. The high level of receptor in endosomes, along with low or undetectable levels in lysosomes , has been interpreted to indicate that newly synthesized lysosomal enzymes are delivered to acidic prelysosomal/endosomal com partments rather than to lysosomes (I). The low pH of the endosomal compartment would cause the ligand-receptor complex to dissociate, and the released lysosomal enzymes could then be packaged into lysosomes while the MPRs could recycle either back to the Golgi and/or to the plasma membrane. It is currently unclear whether the Golgi-derived vesicles containing lysosom al enzyme-receptor complexes fuse with early or late endosomes or with both types of endosomes ( l l Oa). If delivery to early endosomes does occur, the ligand-MPR complex would presumably not dissociate until the complex migrates to late endosomes. This is because significant dissociation of ligand from either receptor requires pH

Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells.

Insulin-like growth factor II effects mediated through insulin-like growth factor II receptors.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

Hematopoietic growth factor receptors.

Effects of partial hepatectomy on hepatic insulinlike growth factor binding protein-1 expression.

Effect of tamoxifen on serum insulinlike growth factor I levels in stage I breast cancer patients.

Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation.

Effect of puberty on insulinlike growth factor I and HbA1 in type I diabetes.

Insulinlike growth factor I: a potent mitogen for human osteogenic sarcoma.

Truncated and native insulinlike growth factor I enhance mucosal adaptation after jejunoileal resection.

Lack of growth hormone effect on insulin-associated suppression of insulinlike growth factor binding protein 1 in humans.

cation-independent mannose-6-phosphate receptors by specific phosphorylated sugars and antibodies induce insulin-like growth factor-II effects.

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

Single-molecule dynamics of site-specific labeled transforming growth factor type II receptors on living cells.

Characterization of type II insulin-like growth factor (IGF) receptors in sheep liver plasma membranes.

Targeting growth factor receptors with fusion toxins.

Epidermal growth factor receptors in the oesophagus.

The nerve growth factor family of receptors.

Subunit promiscuity among hemopoietic growth factor receptors.

Mannose-6-phosphate receptors are increased in primary human thyroid neoplasms.

The effects of infusion of insulinlike growth factor (IGF) I, IGF-II, and insulin on glucose and protein metabolism in fasted lambs.

Mitogenic response of canine fundic epithelial cells in short-term culture to transforming growth factor alpha and insulinlike growth factor I.

Clinical significance of serum insulin-like growth factor-1 (IGF-1) and insulinlike growth factor binding protein-3 (IGFBP-3) in patients with epithelial ovarian cancer.

Angiotensin II: hemodynamic regulator or growth factor?