Perspectives in Diabetes The Insulin Receptor A Multifunctional Protein JERROLD M. OLEFSKY

The insulin receptor is a multifunctional protein encoded by a modular gene. Certain discrete domains within the insulin-receptor structure subserve specific functional properties. In some instances, these discrete domains are encoded by individual exons. This organizational model of the insulin receptor predicts the existence of divergent signaling pathways facilitating specific bioeffects. Some of these signaling pathways are shared with the closely related insulinlike growth factor I receptor (convergent pathways), whereas others are different (divergent). The concept of discrete functional domains also provides several mechanisms whereby inactive insulin receptors (no kinase activity) can inhibit the function of normal receptors. The ability of kinase-inactive insulin receptors to inhibit the signaling function of normal insulin receptors may be an operative mechanism in certain insulin-resistant states. Diabetes 39:1009-16, 1990

T he insulin receptor is a complex multifunctional pro- tein subserving various biological effects. The re- ceptor's organizational structure was firmly estab- lished, based largely on the amino acid sequence

deduced after cloning of the insulin receptor cDNA by Ullrich et al. (1) and Ebina et al. (2). Subsequently. Seino et al. (3) showed that the cDNA was derived from a gene of >I 40,000 M, composed of 22 exons located on chromosome 19. The intron-exon organizational structure of the insulin-receptor gene is shown in Fig. 1.

INSULIN RECEPTOR AS PRODUCT OF MODULAR GENE As rev~ewed by Gilbert (4) and shown by Sudhof et al. (5,6) for the low-density lipoprotein (LDL) receptor, it IS probable

From the \Department of Medicine, Divlsion of Endocrinology and Metabolism, Univers~ty of California, San Diego, La Jolla. and the Veterans Administration Medical Center, San Diego, California.

Address correspondence and reprint requests lo Jerrold M Olefsky, MD, Department of Mediclne, Division of Endocrinology and Metabolism. Univer- sity of California, San Diego, La Jolla, CA 92093

Received for publication 3 May 1990 and accepted 14 May 1990

that the insulin receptor is a modular protein containing sev- eral specific functional domains, some of which are encoded at the gene level by discrete exons. The striking correlation between intron-exon gene structure and encoded functional domains of the LDL receptor (5,6), as well as the similarity of exons in genes that encode related receptoriike proteins (7-1 I ) , argues strongly for the concept that multidomain proteins (e.g., receptor tyrosine kinases) evolved by exon shuffling and sequence duplication events (7-12). Based on current knowledge of insulin-receptor function and the func- tion of other modular proteins, it seems reasonable to pro- pose that specific functional properties of the insulin receptor are subserved by separate domains (Figs. 1 and 2; 5-12). In addition, insofar as discrete linear sequences of the re- ceptor serve as specific functional domains, at least in some cases, these domains may be encoded by specific exons. In this way, the multifunctional insulin receptor can be en- visioned as the product of a modular gene that evolved by the assembly of existing DNA sequences in a cassette-like fashion to encode required protein functions. Over the course of evolution, some degeneration of function at the protein level as well as gene rearrangments have led to overlap of some functions across current intron-exon bound- aries.

The insulin receptor is synthesized as a single-chain pre- cursor polypeptide containing 1343 (6) or 1355 (7) amino acids preceded by a 27-residue NH,-terminal signal se- quence. During processing and transport to the cell surface, the 27-amino acid leader sequence is cleaved, and oligo- saccharide side chains are added at specific glycosylation sites. Two monomers associate to form a dimeric structure, and a cleavage site consisting of four basic amino acids (Arg-Lys-Arg-Arg; positions 720-723) is removed, resulting in discrete a- and p-subunits. These subunits are held to- gether by ap-disulfide bonds to form the mature heterotet- rameric a,p,-insulin receptor. The two identical a-subunits are entirely extracellular and contain either 719 or 731 res- idues, depending on the presence or absence of a 12-amino acid insert (positions 720-731), which arises by alternate splicing of exon 11 into the insulin-receptor mRNA transcript in a tissue-specific manner (13,14). The p-subunit is a 620-

DIABETES, VOL. 39, SEPTEMBER 1990

Signal Insulin binding

I Cysteine Alternatively /Proreceptor Tyrosine

rich spliced exon processing site kinase I - ~ransmembrane . L--y--J FIG. 1. Insulin-receptor gene intronlexon

a-subunit $-subunit structure, depicting 22 exons, their lntronlc I I I I I I I I I I I I I I boundaries, and corresponding encoded regions 0 10 20 30 40 50 60 70 80 90 100 110 120 130 kb of insulin receptor.

residue membrane-spanning protein containing an extra- cellular region of 194 residues, a transmembrane anchoring domain of 23 amino acids, and a 403-residue cytoplasmic extension.

INSULIN-RECEPTOR STRUCTURE AND FUNCTION The idea of specific functional domains within the insulin- receptor molecule (and the concept that at least some of these domains may be encoded by discrete exons) is sup- ported by a substantial amount of experimental data For example, the a-subunits contain a characteristic cysteine- rich region, which is critical to the ligand-binding function (Fig. 1). Sim~lar cysteine-rich regions have been identified in other ligand-binding proteins, e.g., the receptors for LDL, epidermal growth factor (EGF), platelet-derived growth fac- tor (PDGF), and insulinlike growth factor I (IGF-I) (5,6,8,11, and refs. therein). This commonality suggests that cysteine- rich domains provide a unique structural framework con- ducive to high-affinity binding interactions between peptide ligands and the surface-exposed hydrophilic regions of their specific plasma membrane-anchored receptor molecules. Perhaps the intramolecular and intermolecular disulfide bonds in this region form a relatively rigid scaffolding struc- ture, allowing othera-subunit sequences to fold into a ligand- b~nding "pocket." It seems likely that, for this particular receptor function (ligand binding), multiple anatomically separate amino acid sequences participate to form a highly specific binding site that interacts with the insulin molecule, leading to high-affinity association. At least two other regions of the insulin receptor have been proposed as contributors to the binding domain. Yip et al. (15) cross-linked insulin to the receptor and identified a receptor-region (residues 281- 291) amino terminal to the cysteine-rich region that repre- sents at least one contact point for the insulin molecule. Using a different approach, DeMeyts et al. (16) identified a Phe-Phe doublet at residues 88 and 89 as another contact point. It seems likely that these separate regions of the a - subunit (and possibly others) participate with the cysteine- rich framework region to form the tertiary structure, confer- ring a high-affinity binding domain. The interactions between the binding domains of the insulin molecule and the insulin receptor are highly specif~c with very low cross-reactivity to other ligand-receptor systems (e.g., the highly homologous IGF-IIIGF-I receptor system shows 11 % cross-reactivity).

Other functional properties exist within the a-subunit, in addition to ligand binding. Cysteine residues contained within the COOH-terminal region of the a-subunits form di- sulfide bonds with p-subunit cysteine residues linking the a- and (3-subunits. Exon 11 encodes a 12-amino acid insert that is alternately spliced into the insulin-receptor mRNA in a tissue-dependent fashion (13,14). It is also possible that alternate splicing of this exon is developmentally regulated.

Recent studies have shown that this 12-amino acid domain modifies receptor-binding affinity; receptors containing this domain have a twofold to threefold lower binding affinity than those without (14). Finally, it has been reported that insulin receptors aggregate on the cell surface after ligand binding (17,18). It seems possible that, after insulin occupancy, a conformational change is induced in the a-subunits, which exposes regions conducive to interreceptor interactions fa- cilitating aggregation.

Little is known about possible functional properties of the extracellular portion of the p-subunit. Clearly, the cysteine residues in the p-subunit ectodomain participate in disulfide

2nd Messengers , ( K , ;.Yco~~

@ Metobol~c ~ f f e c t s

Cell D ~ v ~ s ~ o n Gene Regu la t~on

FIG. 2. Multifunctional modular insulin receptor. Blocks, known functional domains, some of which are encoded by specific exons. This model illustrates tyrosine klnase properties of ligand-activated lnsulin receptor, including ATP binding, autophosphorylation, and subsequent activation of tyrosine kinase activity. Endogenous protein substrates are shown interacting with discrete domains of insulin receptor, whereon they are phosphorylated on tyrosine residues followed bv dissociation of ~ h ~ s ~ h ~ ~ l a t e d substrate. As theoretical example, idistinct substrates inierai with 2 discrete regions (substrate binding domains) of insulin-receptor COOH-termlnal. Each substrate subserves a separate biological action (metabollc effects vs. mitogenic stimulatlon). Substrate phosphorylation is only one mechanism that fits with this overall model; discrete regions of 0-subunit could interact with other coupling molecules that signal specific actions through non-phosphorylation-related mechanisms.

DIABETES. VOL 39, SEPTEMBER 1990

bonding to the a-subunit. It has been suggested that the extracellular p-subunit region participates in transmitting the insul~n signal through the membrane to the cytoplasmic re- gion of the p-subunit (19). Mutagenesis studies of this region of the p-subunit will be necessary to support this notion.

The 23-amino acid transmembrane region of the p-sub- unit anchors the receptor in the plasma membrane. To a large extent, transmembrane domains can be interchanged between two different surface receptors without impairing the function of either (1 1,20-22). Studies of chimeric recep- tors consisting of extracellular, intracellular, and transmem- brane domains of the EGF, insulin, and IGF-I receptors, in various combinations, have shown that a particular recep- tor's Igand-binding domain can couple with a kinase domain regardless of the origin of the transmembrane region (20- 22). In the chimeric receptors studied, the ligand-binding domain of one receptor can activate the kinase domain of another receptor regardless of which receptor contributes the transmembrane domain. In addition, extensive mutations of the EGF-receptor transmembrane domain did not affect EGF signaling (23). These transmembrane swapping ex- periments and mutagenesis studies suggest that, other than anchoring the receptor in the membrane, the specific se- quences of the insulin-receptor transmembrane domain are not essential for signaling functions.

Because of its intracellular location, the 403-amino acid cytoplasmic extension of the p-subunit must play the critical role in transferring external information (ligand binding) to the cell interior and hence mediate the pleiotropic biological effects of insulin. In addition to standard techniques of pro- tein chemistry, investigators have used the methods of site- directed mutagenesis and have taken advantage of naturally occurring receptor mutants to elucidate functional properties of the p-subunit. At minimum, the cytoplasmic extension of the p-subunit contains a tyrosine kinase domain, several autophosphorylation sites, and a COOH-terminal rich in in- sulin-receptor-specific sequences. When insulin binds to the a-subunit, the tyrosine kinase property of the p-subunit is stimulated, allowing the receptor to autophosphorylate rap- idly on specific tyrosine residues and to become activated as a tyrosine kinase (24-27). The discovery of insulin-stim- ulated tyrosine kinase activity was first made by Kasuga et al. (2829), and this important observation has generated enormous interest in the role of the kinase activity in insulin signaling.

As originally reported by Kasuga et al. (28,29) and con- firmed iri numerous laboratories (24-27 and refs. therein), p-subunit autophosphorylation with subsequent activatron of the k~nase activity is the earliest known signaling event fol- lowing binding of insulin to the extracellular a-subunit. It has been proposed that the activated receptor phosphorylates endogenous protein substrates that mediate insulin's bioef- fects (24-29). Several phosphoproteins, whose tyrosine phosphorylation is enhanced by insulin, have been identi- fied. These putative substrates may be effector molecules, intermediary signaling molecules, or serine kinases involved in a phosphorylation cascade (30). However, although these phosphoproteins have been identified, no known physiolog- ical function for any insulin-stimulated substrate has been documented. As an alternative to the substrate phosphor- ylation concept, it is possible that autophosphorylation or

some other sequelae of ligand binding induces a confor- mational change, allowing the receptor to interact produc- tively with other cellular components (e.g., G proteins, phos- pholipases) to signal bioeffects independent of substrate phosphorylation. Insulin-mediated stimulation of phospholi- pase activity has been documented, and occupied recep- tors may interact with G protein-like signaling molecules to propagate the insulin-action cascade, particularly for insu- lin's acute metabolic effects. These mechanisms are not mu- tually exclusive, because substrate phosphorylat~on may mediate some insulin actions with noncovalent interactions mediating others.

Tyrosine kinase activity is a property common to many polypeptide hormone-receptor systems (e.g., the receptors for IGF-I, EGF, PDGF, and the colony-stimulating factor [CSF]), and these different receptor tyrosine k~nases share common structural features (8). By comparing the sequence of the insulin-receptor p-subunit to that of the other receptor tyrosine kinases and to tyrosine kinase-active protoonco- genes, it can be seen that the kinase region is the most highly conserved portion of these molecules (7-1 1 ) . As a result, sequence homologies revealing certain structure and function relationships can be identified. Binding of ATP is an essential function for all protein kinases, and an ATP-binding site can be readily identified in the p-subunit comprising the Gly-X-Gly-XX-Gly sequence located at residues 991-996 (from A. Ullrich's numbering system). In addition, a char- acteristic lysine-residue COOH-terminal to the Gly-X-Gly-XX- Gly sequence can be identified at position 1018; this lysine is thought to form a salt bridge, with the p-phosphate of ATP facilitating tight ATP binding. The presence of this defined ATP-binding domain has facilitated detailed studies of the role of autophosphorylation/kinase activity in receptor func- tions. By site-directed mutagenesis, point mutations have been inserted into the insulin-receptor cDNA modifying the lysine at position 1018. Via this approach to abolish ATP binding, lysine 101 8 has been modified to alanine (31,32), arginine (33), or methionine (33). In all cases, after trans- fection and overexpresslon in host cells, a receptor devoid of tyrosine kinase activity was produced. Predictably, this mutant demonstrated the ATP-binding requirement for sub- sequent kinase activity; however, the major importance of this receptor mutant was that it allowed studies of the role of kinase activity in insulin receptor biology.

RECEPTOR KINASE ACTIVITY The results obtained with the lysine 1018-mutated receptors have been consistent (31-33). In all cases, when overex- pressed in various cell types, these receptors display an absence of insulin-stimulated autophosphorylation and ki- nase activity and fail to mediate any of insulin's biological effects (31-33). Neither insulin nor various anti-insulin-re- ceptor antibodies, which normally display agonistic prop- erties, are able to generate biological signaling through this kinase-negative mutant receptor (31-33). Taken together, these results provide a strong argument in favor of the role of insulin-receptor autophosphorylationikinase activity in me- diating some, i f not all, of insulin's biological effects. The role of autophosphorylation in the activation of the kinase has been investigated by mutating the key tyrosine phosphory- lation sites. On the basis of earlier studies, it has been shown

DIABETES, VOL 39, SEPTEMBER 1990 1011

that the three tyrosine residues located at positions 1146, 11 50, and 11 51 are largely phosphorylated together and are the most important sites associated with an active receptor kinase state (34,35). These residues have been mutated singly or in various combinations in several studies. In gen- eral, ~t has been found that autophosphorylation-site muta- tion leads to a reduction in receptor kinase activity and com- promises biological signaling (36); however, the results from this class of mutations have not been as consistent or une- quivocal as with the ATP-binding mutants. For example, De- bant et al. (37) reported that substitution of tyrosines 1150 and 1151 with phenylalanine abolishes insulin stimulation of glucose transport but not m~togenic signaling. Modification of autophosphorylation sites may not be as damaging to kinase funct~on as deletion of ATP binding and may be qual- itatively different. Other receptor constructs without insulin- stimulated kinase activity, because of deletions or trunca- tions, also show loss of biological signaling (36,38,39).

KINASE-INACTIVE RECEPTOR INHIBITION OF FUNCTION OF NORMAL RECEPTORS Another interestng observation has emerged from studies of the ATP-binding-defective insulin receptor. Although the receptor does not signal any of insulin's biological effects, it does not behave as an inactive molecule or neutral by- stander with respect to normal insulin act~on. When ex- pressed in rodent cells that display their own endogenous receptors, the kinase-inactive receptors inhibit the function of the native receptors (31-33). The dose-response curves for insulin stimulation of biological functions (glucose trans- port, glycogen synthesis. S6 kinase, c-fos induction, and thymidine uptake) are depressed andlor shifted to the right compared with parental cells. Thus, overexpression of the kinase-inactive receptors produces a state of cellular insulin resistance.

How do the kinase-~nactive receptors inhibit insulin sig- naling through the normal native receptors?There are at least three possibilities (40,41). 1 ) Rodent-human hybrid recep- tors could form, consisting of a normal rodent ap-half and a kinase-inactive human up-half. If p-subunit autophospho- rylation within an insulin-receptor heterotetramer occurs by a trans rather than a cis mechanism, or if two normal p- subunits are otherwise necessary to confer signaling com- petency, such hybrid receptors would be inactive (10). 2) It is possible that multimeric aggregation of receptors at the cell surface is necessary to generate a biological signal. If kinase-inactive receptors participate in this aggregation, they could render the aggregate inactive. 3 ) Kinase-inactive receptors may compete with normal receptors for endoge- nous protein substrates or other limiting downstream sig- naling molecules.

When a receptor with alanine substituted for lysine at po- sition 1018 (AK 1018) was transfected into the rat fibro- blast background, all of the measured biological effects of insulin were inhibited by the presence of the AK 1018 re- ceptors (31-33, 40, 41). These cells also expressed many endogenous IGF-I receptors, which mediatemany biological effects common to the insulin-receptor system. In the AK 1018 cell lines, IGF-I remains fully capable of stimulating glucose transport, whereas the AK 1018 insulin receptors inhibit IGF-I stimulat~on of mitogenesis (40,41). In these AK

1018 cells, only a small fraction of the endogenous rat insulin receptors appeared to form hybrids with the human AK 101 8 polypeptide (40). Because the expression of endogenous receptors is relatively low (3000-5000) in these cells, how- ever, these data are difficult to quantitate. It could be dem- onstrated more clearly that only a small percentage (-10- 20%) of the endogenous IGF-I receptors form hybrids with the AK 101 8 receptor (hybrid IGF-IIAK 1018 heterotetramers; 42), yet IGF-I effects on mitogenesis are fully inhibited (41 ). These results indicate that formation of some inactive hybrid receptors can occur, but this is insufficient to explain the inhibition fully. Whether inactive aggregates occur on the cell surface is unknown, but because AK 1018 insulin re- ceptors inhibit IGF-I-receptor mitogenic signaling, the oc- currence is unlikely. The receptors for insulin and IGF-I do not seem to aggregate. Even if they did, mitogenic action with normal glucose-transport stimulation should not be in- hibited.

Another hypothesis to explain the inhibition involves the concept of substrate competition. According to this line of reasoning, activated insulin receptors phosphorylateendog- enous substrates. With this model, there must be some phys- ical association between the substrate and the cytoplasmic portion of the p-subunit (i.e., association of substrate with a substrate-binding site). If so, then perhaps substrate phos- phorylation participates in dissociation of the substrate-re- ceptor complex, allowing the substrate to propagate the in- sulin-signaling cascade. Because the AK 101 8 receptor has only a single point mutation at the ATP-binding site, the pu- tative substrate-binding sites should be unperturbed. Sub- strates could still interact with the AK 1018 receptor but only in a futile way, because they cannot be phosphorylated. Therefore, the overexpressed kinase-inactive receptors would interact nonproductively with endogenous substrates (or other signaling molecules), effectively titrating them away from the normal native receptors (Fig. 3A). The observation that lnsulin cannot stimulate endogenous substrate phos- phorylation in AK 1018 cells is consistent with this mecha- nism. Ellis et al. (43) showed that a membrane-anchored intact p-subunit, with the extracellular insulin-binding domain deleted, is active as a tyrosine krnase and associated with constitutively elevated rates of ligand-independent glucose transport. When this membrane-anchored p-subunit was mutated at tyrosines 1150 and 1151, no constitutively acti- vated tyrosine kinase or glucose-transport activity was ob- served. The 1150-1 151 construct was markedly inhibitory to normal insulin s~gnaling of glucose transport by the native receptors. Because this membrane-anchored 11 50-1 151 mutant was mostly localized to intracellular membranes of the endoplasmic reticulum (43), whereas most of the native receptors were localized to the plasma membrane, this find- ing argues for a mechanism of inhibition unrelated to direct interaction of the mutated receptor with the native receptor (this mitigates against hybrid hamster-human receptors and impaired aggregation as operative mechanisms for this re- ceptor construct) and supports the concept of substrate competition as the cellular mechanism of inhibition. These possible inhibitory mechanisms are not mutually exclusive; it is possible that inactive hybrid receptors form and that these and the homodimeric kinase-inactive receptors com- Pete with normal receptors for substrates.

1012 DIABETES, VOL. 39. SEPTEMBER 1990

In8ulln receptor IGF-I receptor

A nY 1-l

A. 6. C. D. E.

FIG. 3. A: mechanlsms of inhibition. Kinase-inactive insulin receptors inhibit function of normal insulin receptors when expressed in same cell. One mechanism to explain this form of dominant negative mutation is substrate competition hypothesis, whereby substrates (a) bind to kinaseinactive insulin receptors; because they cannot be phosphorylated, they do not dissociate, making them relatively unavailable to normal receptors. This lowers effective substrate concentration available to normal receptors, reducing their ability to signal biological effects. Competition for key substrate molecules is only 1 mechanism fitting this model. If coupling molecules other than substrates are involved in insulin slgnaling, then competition for these molecules could occur. Inhibition could also occur by formation of inactive hybrid receptors or inactive cell surface receptor aggregates; a combination of these mechanisms is also possible. 8: divergent and convergent signaling mechanisms for insulin and IGF-I receptors. Each receptor has divergent signaling mechanisms for specific bioeffects. Between receptors, some signaling pathways can be entirely separate (divergent), and others can be common or shared (convergent).

In this way, kinase-negative receptors are inactive by themselves and inhibit the function of the normal receptors, behaving as a dominant negative mutation (44). This form of a dominant negative mutation may explain why patients who inherit genetic defects in insulin receptors are more insulin resistant when they are heterozygous for kinase-in- active receptors than when they are heterozygous for other kinds of mutations (J. Kusari, Y. Takata, O.G. Kolterman, E.N. Hatada, and J.M.O., unpublished observations). Kinase-in- active insulin receptors are a feature of various human in- sulin-resistant states. The mechanisms can involve acquired modifications as in common non-insulin-dependent diabetes mellitus (45-47) or inherited structural abnormalities in pa- tients with unusual forms of genetic insulin resistance or d~abetes (48). Kinase-inactive insulin receptors may worsen the insulin-resistant state by inhibiting the function of normal receptors and thus amplifying their own negative signaling effects.

DIVERGENT SIGNALING PATHWAYS Considering the insulin receptor and the closely related IGF- I receptor as multifunctional multidomain proteins leads to

some important predictions about the biology of hormone signaling. For example, envisioning multiple signaling do- mains within the p-subunit (Figs. 1 and 2) leads to the con- cept of multiple divergent signaling pathways (Fig. 38) . The signaling domains within the p-subunit could involve multiple binding sites for endogenous protein substrates that become phosphorylated on tyrosine residues by the activated re- ceptor kinase. These phosphorylated substrates would then be released to participate in downstream signaling as direct effector units or coupling molecules. For example, a sub- strate could be a serine kinase that participates in propa- gating the phosphorylation cascade. Indeed, certain serine kinases such as microtubule-associated protein II kinase, raf, and S6 kinase have been identified as phosphorylation targets of receptor tyrosine kinases (30,49). Substrate phos- phorylation is not the exclusive mechanism that fits this model. Autophosphorylation or some other conformation change induced by ligand binding could allow domains of the receptor to interact productively with other signaling mol- ecules, e.g., G proteins and phospholipases. With this par- adigm, the different signaling domains of the p-subunit could interact with different downstream molecules. Alternatively, a combination of receptor kinase and non-phosphorylation- dependent mechanisms might exist.

Several lines of evidence suggest that divergent signaling pathways are present for insulin action and that this diver- gence begins at the receptor level. Studies of an insulin receptor truncated by 43 amino acids at the COOH-terminal (ACT receptor) support this line of reasoning (50-52). The logic in constructing this mutant was that the COOH-terminal represents a region poorly homologous to the IGF-I or EGF receptor and, thus, is rich in insulin-specific sequences (53). Therefore, its deletion might adversely affect those actions of the insulin receptor that are most insulin specific. The ACT receptor is synthesized and processed normally (50). It undergoes insulin-stimulated autophosphorylation and can be act~vated in a relat~vely normal fashion as a kinase di- rected toward various exogenous and endogenous sub- strates (51). Although normally activated as a kinase, the ACT receptor signals insulin's metabolic effects (glucose transport and glycogen synthase stimulation) poorly (51). Thus, removal of the COOH-terminal leaves the kinase intact, but it deletes structural information crucial for metabolic sig- naling. A discrete binding site might exist in the COOH- terminal whose function is necessary for metabolic action. Alternatively, the COOH-terminal could be the domain of the receptor that interacts in a noncovalent way with subsequent cell components necessary for metabolic action. This lack of biological signaling was not true for all insulin effects. The truncated receptor was supernormal in its abllity to mediate insulin's mitogenic effects (thymidine uptake). Thus, the dose-response curve for insulin-stimulated thymidine uptake was shifted markedly to the left in the ACT cells compared with the parental cell line and transfectants expressing sim- ilar numbers of wild-type human insulin receptors (52). In a functional sense, the truncation converted the insulin recep- tor from a largely metabolic receptor to a growth or mitogenic receptor. These studies clearly show the divergence of var- ious insulin-signaling pathways and indicate that the COOH- terminal contains positive information critical to metabolic signaling and that the absence of the COOH-terminal en-

DIABETES. VOL 39. SEPTEMBER 1990

hances the mitogenic or growth-promoting activities intrinsic to the insulin receptor.

The concept of domain-specific signaling functions intrin- sic to the insulin receptor is also supported by the obser- vations that, within a single cell type, insulin resistance can exist for one insulin action while another action is normal (54). Debant et al. (37) reported studies of a mutant insulin receptor in which two of the tyrosine autophosphorylation sites (1 150 and 11 51) were changed to phenylalanine. They found that this receptor did not signal stimulation of glucose transport or glycogen synthesis but was fully capable of stimulating thymidine uptake. Similarly, Wilden et al. (55) studied an insulin receptor in which one of the autophos- phorylation sites (1 146) was mutated to phenylalanine. This receptor retained normal metabolic signaling, whereas mi- togenic signaling was severly compromised. Again, a mu- tation at the receptor level was able to adversely influence one insulin-action pathway but not another.

Finally, the effects of the AK 1018 receptor on IGF-I-re- ceptor signaling are also supportive of divergent pathways (40,41). IGF-I can stimulate metabolic effects normally in AK 1018 cells (40), but it is almost completly inhibited with re- spect to mitogenic stimulation (41). The lack of mitogenic stimulation in AK 1018 cells is specific for insulin and IGF- I, because serum and EGF stimulate mitogenesis normally in these cells. In the AK 1018 cells, insulin does not stimulate glucose transport whereas IGF-I does, indicating divergence of this signaling pathway between the two receptors (Fig. 38). Because mitogenic signaling by both receptors is in- hibited in these cells, insulin and IGF-I receptors may share the same signaling mechanisms for mitogenesis, indicating a convergent pathway (Fig. 38). Thus, for a single receptor, signaling pathways may be divergent or convergent, and between heterologous receptors, pathways may also be di- vergent or convergent.

RECEPTOR ITINERARY Insulin binding initiates receptor internalization and subse- quent sorting of the internalized receptor through recycling or degradative pathways (56). Because insulin-receptor in- ternalization is markedly accelerated after ligand binding, it seems likely that insulin binding to the receptor induces a functional or conformational change, which mediates en- docytotic receptor uptake. What regions of the insulin re- ceptor contain the information necessary for this function? Monomeric receptor tyrosine kinases, e.g., the EGF receptor, dimerize at the cell surface before their internalization via coated pits (1 1). The insulin-receptor heterotetramer is anal- ogous to a preexisting dimeric structure; although insulin receptors aggregate after ligand binding, the importance of aggregation for the internalization event has not been dem- onstrated (17,18). In any event, after initial ligand binding, insulin-receptor complexes are internalized by absorptive endocytosis into target cells. After internalization, Insulin dis- sociates from its receptor within the endocytotic vesicle be- cause of a drop in vesicular pH (acidification); then the in- tracellular pathways for insulin and the receptor segregate (5657). The insulin is degraded to insulin intermediates and then to low-molecular-weight products that are eventually reutilized or released. Intact insulin can also be released from the cell by a regulated and tissue-specific process lermed reboendocytosis (54,56). The fate of the internalized

receptor can include recycling back to the plasma mem- brane, sequestration within the cell, and degradation. Thus, the entire process can be named the insulin-receptor itin- erary and serves to clear bound Insulin from the cell surface and deliver the ligand to intracellular sites of degradation. At the same time, efficient reutilization of the receptor is maintained in most cell types by the recycling process. Dur- ing its intracellular itinerary, the receptor may also mediate biological functions or have its own functions modified (by serine phosphorylation, oxidation, acylation, or other cova- lent modifications) at intracellular sites, and endocytosis may be involved in termination of certain signaling events.

Specific aspects of insulin-receptor structure or function are critical to the steps in this itinerary. For example, recep- tors made kinase inactive by modification of lysine 101 8 do not undergo insulin-mediated endocytotic uptake (31,3258). These studies suggest that autophosphorylation or some aspect of receptor kinase activity is important for internali- zation. Analogously, an ATP-binding-defective mutant EGF receptor also fails to undergo ligand-mediated internalization (59). Note that the constitutive (basal) rate of insulin- or EGF- receptor internalization is unaffected by rendering receptors kinase inactive (58,59). Mutation of tyrosine autophosphor- ylation sites generates an insulin receptor with markedly diminished internalization properties (55,60). Other studies in liver cells have shown, however, that when receptor auto- phosphorylation is diminished by depleting cells of ATP, re- ceptor internalization can still be demonstrated (61). In liver cells, mutated kinase-inactive receptors can still endocytose (62). Note, however, that major differences in the organi- zational structure of receptor internalization exist among dif- ferent cell types with respect to the requirement for coated pit internalization, noncoated pit internalization, macroen- docytosis, and so on (1 7,1836). Thus, the precise role of autophosphorylation/kinase activity in receptor endocytosis has not been fully elucidated, and its importance might de- pend on what unique mechanisms and structures particular cell types use for internalization. Perhaps binding of ATP to the p-subunit rather than autophosphorylation or kinase ac- tivity is important for internalization.

Other p-subunit domains also participate in internalization. Chen et al. (63) showed that a unique NPXY sequence located in the immediate submembranous region of the LDL-receptor cytoplasmic extension mediates LDL-receptor endocytosis. They also pointed out that this sequence is represented in many other cell surface receptors. For the EGF receptor, three NPXY sequences are localized to the COOH-terminal; a 213-residue COOH-terminal (to position 973) deletion, containing all three NPXY sequences, pro- duced a noninternalizing EGF-receptor mutant (64). Deletion of only 154 residues (to position 1022), also removing all three NPXY sequences, resulted in only a modest internal- ization defect (64). Thus, EGF receptors missing all NPXY sequences display substantial rates of ligand-induced in- ternalization, indicating no absolute requirement of these sequences for endocytosis (64). Thies et al. (65) deleted a 22-residue submembranous portion of the insulin receptor encoded by exon 16 (which includes the insulin-receptor NPXY sequence). Studies of this deletion-mutant receptor show that it does not undergo insulin-mediated internaliza- tion, even though the kinase properties are unaffected. Whether NPXY residues or other structural features of the

1014 DIABETES, VOL. 39, SEPTEMBER 1990

deletions abrogate endocytosis is unknown; point mutations to modify single amino acids within this sequence will be necessary to determine which features of the deleted do- mains are important for internalization.

Little is known about the sorting signals influencing the remainder of the receptor itinerary; however, Riedel et al. (66) created chimerlc receptors consisting of I ) the EGF- receptor ectodomain and the insulin-receptor cytoplasmic domain (EIR) and 2) the insulin-receptor ectodomain and the EGF-receptor cytoplasmic domain (IER). They found that both chimeras bind the appropriate ligand, signal kinase activat~on, and internalize. After endocytosis, the IER is rap- idly degraded (like wild-type EGF receptors), whereas the EIR is mostly recycled (like wild-type insulin receptors). This indicates that the cytoplasmic domain contains the sorting signals that govern the fate of the receptor as it transverses its itinerary (66). Other studies have shown that deletion of the COOH-terminal 43 amino acids of the insulin receptor does not impair internalization, recycling, retroendocytosis, receptor turnover rate, downregulation, or ligand degrada- tion (50). These studies are important because the p-subunit is exposed to the cytoplasm after endocytosis, and they indicate that targeting signals are not contained within the COOH-terminal. These data focus the search for sorting sig- nals on the remainder of the cytoplasmic p-subunit region. It can be speculated that the insulin-receptor region between the transmembrane domain and the beginning of the kinase domain may contain the sorting signals that govern the fate of the receptor as it transverses its intracellular itinerary.

INSULIN-RECEPTOR-DOMAIN INTERACTIONS The belief that every functional property of the insulin re- ceptor derives from a specific linear doma~n is clearly an oversimplification, because some properties require a ter- tiary structure involving the interaction of anatomically sep- arate amino acid sequences to confer a particular function. In addit~on, a situation with one domain influencing the func- tional properties of another can be envisioned. For example, when unoccupied, the a-subunit exerts a tonic inhibitory in- fluence over the cytoplasmic tyrosine kinase activity. After ligand binding, this inhibitory effect is released, leabing to activation of the receptor kinase properties. When the COOH-terminal of the insulin receptor is truncated (52) or the COOH-terminal tyrosine residues (1316, 1322) are mod- ified (67), the resulting receptor mutants display enhanced insulin-induced mitogenic signaling properties. This sug- gests that the receptor COOH-terminal, particularly tyrosines 1316 and 1322, normally exerts an inhibitory influence over mitogenic signalrng by the insulin receptor. This is analogous to other receptor tyrosine kinases, where inhibitory effects of COOH-terminal domains have been demonstrated (1 1). Finally, the 12-amino acid insert at the COOH-terminal of the a-subunit encoded by exon 1 1 exerts a modulating effect on the affinity of the receptor for insulin (14). It is likely that other influences of one receptor domain on another will be identified.

SUMMARY The insulin receptor is the product of a modular gene whose exons, at least in part, may have been assembled during evolution by bringing together blocks of DNA sequences from other genes to encode specif~c functions. The resulting

protein product, the ~nsulin receptor, is a multifunctional mol- ecule containing discrete domains, some of which subserve specific functional properties. This model predicts that fo- cused structural alterations in the insulin receptor will modify specific functional properties, leaving others unaffected. This prediction is supported by numerous studies of chimeric receptors, naturally occuring receptor mutants, and recep- tors created by site-directed mutagenesis. This organization allows the insulin receptor to participate in divergent biolog- ical signaling pathways and predicts that, insofar as highly homologous domains are shared by insulin receptors and other receptors, convergent signaling pathways will exist. It is also possible that altered domains of one insulin receptor can interfere with the function of the corresponding normal domain of another receptor in a dominant way, leading to impaired cellular functioning. With available techniques, knowledge of insulin-receptor structure should rapidly ad- vance and reveal many of the molecular and biochemical details of insulin-receptor function. This will not only improve understanding of insulin action but may also lead to new insights and therapeutic approaches in states of altered in- sulin action such as certain forms of diabetes.

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