Insulin Signalling

Cell. Signal. Vol. 11, No. 8, pp. 563–574, 1999 ISSN 0898-6568/99 $–see front matterCopyright 1999 Elsevier Science Inc. PII S0898-6568(99)00025-X

TOPICAL REVIEW

Insulin Signalling: Metabolic Pathways andMechanisms for SpecificityFredrik H. Nystrom and Michael J. Quon*

Hypertension-Endocrine Branch, National Heart, Lung and Blood Institute,National Institutes of Health, Building 10, Room 8C-103, 10 Center Drive MSC 1754,

Bethesda, MD 20892-1754, USA

ABSTRACT. Biological actions of insulin are mediated by the insulin receptor, a member of a large family ofreceptor tyrosine kinases (RTK). Signal transduction by the insulin receptor follows a paradigm for RTK signal-ling. Many intracellular signalling molecules contain multiple modular domains that mediate protein-protein in-teractions and participate in the formation of signalling complexes. Phosphorylation cascades are also a promi-nent feature of RTK signalling. Distal pathways are difficult to dissect because branching paths emerge fromdownstream effectors and several upstream inputs converge upon single branch points. Thus, insulin action isdetermined by complicated signalling networks rather than simple linear pathways. Interestingly, many signallingmolecules downstream from the insulin receptor are also activated by a plethora of RTKs. Therefore, mechanismsthat generate specificity are required. In this review we discuss recent advances in the elucidation of specific met-abolic insulin signalling pathways related to glucose transport, one of the most distinctive biological actions ofinsulin. We also present examples of potential mechanisms underlying specificity in insulin signalling includinginteractions between multiple branching pathways, subcellular compartmentalization, tissue-specific expressionof key effectors and modulation of signal frequency and amplitude. cell signal 11;8:563–574, 1999. 1999Elsevier Science Inc.

KEY WORDS. Signal transduction, Signal specificity, GLUT4, Insulin resistance

INTRODUCTION discuss a number of potential mechanisms for achievingspecificity in insulin signalling that are illustrated by recentInsulin is an essential peptide hormone that regulates me-studies.tabolism, growth, and differentiation. Biological actions of

insulin are initiated when insulin binds to its cell surface re-ceptor. The propagation of information from the insulin re-

INSULIN SIGNALLINGceptor culminates in diverse effects such as increased glu- FOLLOWS A PARADIGM FOR RTKscose transport, mitogenesis, and regulation of enzymatic Ligand Binding and Receptor Dimerizationpathways (Fig. 1). The insulin receptor belongs to a family

Signal transduction by an RTK is initiated by the specificof ligand-activated receptor tyrosine kinases (RTK) that in-binding of a ligand to the extracellular portion of its cog-cludes receptors for a variety of growth factors [1–5]. Inter-nate cell surface receptor. Ligand binding to monomeric re-estingly, many post-receptor signalling events are shared inceptors (e.g., EGF and PDGF receptors) results in receptorcommon by a multitude of different RTKs. Thus, one of thedimerization, a necessary first step in RTK activation [4, 6,central puzzles in the field of signal transduction is to under-7]. Some RTKs such as the insulin receptor and IGF-1 re-stand how signal specificity is achieved after the binding ofceptor exist in dimeric form even in the absence of ligand.a ligand with its cognate receptor. In this review, we willFor example, the mature insulin receptor consists of two ex-briefly outline how insulin receptor signalling follows a gen-tracellular a-subunits and two transmembrane b-subunitseral paradigm for RTK signal transduction. Particular em-joined by disulfide bonds that constitute an ab heterodimerphasis will be given to pathways related to the stimulation[8]. In the absence of insulin, the a-subunit of the insulin re-of glucose transport since this is an important physiologicalceptor exerts tonic inhibitory influence over the tyrosine ki-action of insulin and is a metabolic function that distin-nase contained in the intracellular portion of the b-subunitguishes the insulin receptor from other RTKs. We will alsoof the receptor [9, 10]. When insulin binds to specific re-gions of the a-subunit, a rapid conformational change in

*Author to whom all correspondence should be addressed. Tel: (301) 496- the receptor results in activation of the tyrosine kinase do-6269; Fax: (301) 402-1679; E-mail: [email protected] 7 January 1999; and accepted 9 February 1999. main [11, 12].

564 F. H. Nystrom and M. J. Quon

FIGURE 1. Insulin signal transduction follows a paradigm for receptor tyrosine kinase signalling.

Receptor Autophosphorylation and (SH2) domains on downstream molecules. SH2 domainsActivation of Intrinsic Tyrosine Kinase are protein domains of ,100 amino acids that share homol-

ogy with a noncatalytic region of the src proto-oncogeneThe kinase region of all RTKs shares substantial homologyproduct. Many molecules involved with RTK signallingin both the ATP binding site and the catalytic domain [4].such as phosphatidylinositol 3-kinase (PI3K), growth factorUpon ligand binding and receptor dimerization, the kinasereceptor bound protein 2 (GRB-2), SH2-containing phos-domain in one half of the dimer phosphorylates cytoplasmicphatase-2 (SHP-2), GTPase activating protein (GAP), phos-tyrosine residues on the other half of the receptor dimer.pholipase C-g (PLC-g), and others, contain SH2 domains.This mutual transphosphorylation event is known as recep-Motifs defined by the three amino acid residues on thetor autophosphorylation and results in a large increase inC-terminal side of the phosphotyrosine residue provide speci-the catalytic activity of the receptor. In the non-phosphory-ficity for interaction with particular SH2 domains [16,17].lated state, the so-called “activation-loop” in the distal

The phosphotyrosine sites that engage specific SH2 do-b-subunit of the insulin receptor occludes the catalytic sitemains of various signalling molecules have been wellso that access of ATP and substrates are blocked. Upon au-mapped for the EGF and PDGF receptors [18,19]. Althoughtophosphorylation of tyrosine residues at positions 1158,the autophosphorylated insulin receptor can also directly1162, and 1163 in the activation loop, this region swingsinteract with SH2 domains in molecules such as PI3K,out of the catalytic site and is stabilized in a conformationSHP-2 and GAP [20–22], these interactions are not criticalthat gives unrestricted access to ATP and substrates [13–15].for insulin signalling. Instead, there are a number of sub-strates for the insulin receptor tyrosine kinase such as insu-

Tyrosine Phosphorylation of Cellular Substrates lin receptor substrate-1 (IRS-1), IRS-2, -3, -4, Shc, andand Recruitment of Distal Signalling Molecules GAB-1 that provide an interface between the insulin recep-

tor and downstream SH2-domain containing signallingActivation of the RTK leads to tyrosine phosphorylation ofmolecules [23–29]. The members of the IRS family sharecellular substrates that propagate signalling. In addition, re-several features in common including an NH2-terminalceptor autophosphorylation enables the RTK to engage di-pleckstrin homology (PH) domain and phosphotyrosinerectly other signalling molecules via interactions between

phosphotyrosine motifs on the receptor and src homology-2 binding (PTB) domain that are important for mediating in-

Insulin Signalling 565

rosine motifs. SH3 domains bind with high affinity to par-ticular proline rich sequences [4]. Some SH2 domain con-taining proteins (e.g., SHP-2, PLC-g) are effector moleculesthat possess intrinsic catalytic activity that is regulated byinteractions of the SH2 domain with phosphotyrosine mo-tifs on other proteins (e.g., IRS-1). These SH2 domains alsohelp localize signalling molecules to particular compart-ments within the cell. Other SH2/SH3 domain containingproteins (e.g., GRB-2, Nck, and the p85 regulatory subunitof PI3K) function as adaptor proteins and have no intrinsiccatalytic activity. These adaptors mediate formation of spe-cific signalling complexes via simultaneous interactions ofFIGURE 2. Insulin receptor substrate (IRS) family members.

Human IRS-1 (hIRS-1 [162]), mouse IRS-2 (mIRS-2 [24]), multiple SH2/SH3 domains on the adaptor protein withmouse IRS-3 (mIRS-3 [34]), human IRS-4 (hIRS-4 [29]) and both upstream and downstream signalling molecules. Acti-human GAB-1 (GAB-1 [26]) share common features including

vation of Ras and PI3K, two major effectors of pathwaysPH and PTB domains as well as multiple phosphotyrosine motifsshared by a number of growth factor receptors including thethat bind to SH2 domains (numbering of amino acid residues for

these domains are indicated along the top of the figures). YXXM insulin receptor, fit this latter pattern. For example, GRB-2motifs bind to the SH2 domains of the p85 regulatory subunit is normally pre-bound to the guanine nucleotide exchangeof PI3K. factor SOS (two SH3 domains of GRB-2 bind proline rich

regions of SOS). When phosphotyrosine motifs on IRS-1 orShc bind to the SH2 domain of GRB-2, the pre-bound SOSteractions with the insulin receptor (Fig. 2) [23, 24, 26, 29–catalyzes the exchange of GTP for GDP on Ras leading to32, 34, 162]. In particular, the PTB domain binds to theits activation. Similarly, the p85 regulatory subunit of PI3Kphosphorylated tyrosine 960 in the NPEY motif of the jux-is normally pre-associated with the p110 catalytic subunit.tamembrane region of the insulin receptor b-subunit [23,Upon insulin stimulation, phosphorylated YXXM motifs on30, 31, 33]. The COOH-terminal portion of IRS proteinsIRS proteins engage the SH2 domains of p85 leading to ac-contain multiple tyrosine containing motifs that undergotivation of p110 (for reviews see [38, 39]).phosphorylation by the insulin receptor and serve as dock-

ing sites for SH2-domain containing proteins. The numberand sequence of these phosphotyrosine motifs is relatively Downstream Phosphorylation Cascadeswell conserved between the various IRS proteins [32]. For

Distal RTK signalling pathways are difficult to dissectexample, multiple YXXM motifs that are known to bind tocleanly because numerous branching pathways begin tothe SH2 domains in the p85 regulatory subunit of PI3K areemerge from single effectors. In addition, multiple upstreampresent in all IRS family members. Thus, phosphorylatedinputs often converge upon single branch points. Further-IRS proteins can mediate the formation of signalling com-more, the presence of negative feedback mechanisms addsplexes consisting of several SH2-domain containing mole-to the complexity of signal transduction. However, somecules. Although IRS proteins share a number of similarities,distal signalling mechanisms such as phosphorylation cas-there are also significant differences between each familycades are shared in common by many growth factors includ-member suggesting that these molecules do not have com-ing insulin. For example, Ras directly activates Raf, a serine/pletely redundant functions. For example, IRS-3 is ,50%threonine kinase that phosphorylates and activates MEKshorter than IRS-1 and -2 and, unlike IRS-1 and -2, doesthat in turn phosphorylates MAP kinase which can thennot contain a phosphotyrosine motif predicted to bind tophosphorylate transcription factors such as Elk-1, leading tothe SH2 domain of GRB-2 [25, 34]. Furthermore, the phe-induction of early immediate genes such as the protoonco-notype of transgenic mice homozygous for null alleles ofgenes c-jun and c-fos. Insulin signalling downstream fromIRS-1 is distinct from the phenotype of IRS-2 knockoutPI3K also involves serine/threonine phosphorylation cas-mice [35–37]. IRS-1 knockout mice are mildly insulin resis-cades. For example, phospholipid products generated by PI3Ktant but do not develop diabetes while IRS-2 knockoutactivate phosphoinositide dependent kinase-1 (PDK1) bymice have both insulin resistance and severe pancreaticbinding to its PH domain. PDK-1 phosphorylates T308 in theb-cell defects leading to abnormal insulin secretion and theregulatory region of Akt (another serine/threonine kinase)development of diabetes. Thus, the various IRS familycontributing to activation of Akt, that in turn phosphorylatesmembers most likely have some overlapping functions, butand inactivates glycogen synthase kinase-3 (GSK-3), leadingare clearly not completely interchangeable.to activation of glycogen synthase [40–42].

Signalling Proteins Containing SH2 and SH3 DomainsProtein Tyrosine PhosphatasesMany signalling molecules downstream from RTKs andAnother aspect of regulation common to RTK signalling istheir substrates contain SH2 and/or SH3 domains that me-the dephosphorylation of RTKs and their substrates by pro-diate protein-protein interactions. As mentioned above,

SH2 domains interact specifically with phosphorylated ty- tein tyrosine phosphatases (PTPases). The number and di-


versity of PTPases rival that of the RTKs [43, 44]. The PTPs where it acts as a facilitative transporter to enhance entryof glucose into the cell [67–70]. This redistribution ofare subdivided into a family of nontransmembrane proteins

containing a single catalytic PTPase domain and a family of GLUT4 is due largely to an effect of insulin to increase therate of exocytosis of GLUT4, although insulin may also havetransmembrane receptor-like PTPases that typically con-

tain tandem PTPase domains. The transmembrane PTPases a minor effect to decrease endocytosis of GLUT4 [71–73].Elucidation of metabolic insulin signalling pathways hashave been further categorized into eight groups based on

shared structural features of various extracellular domains been challenging for several reasons. First, although muscle[44]. The large number of PTPases suggests that each and adipose tissue normally express high levels of GLUT4PTPase plays a specific role in modulating signalling by and are extremely responsive to insulin, the ability toRTKs [45]. PTPases such as SHP-2 contain SH2 domains transfect recombinant DNA or apply other molecularthat confer specificity, while the receptor-like PTPases have methods in these terminally differentiated tissues has beenextracellular domains that presumably interact with specific limited. Second, tissue culture models such as 3T3-L1 adi-ligands. Subcellular localization of particular PTPases may pocytes, L6 myocytes, or C2C12 cells that are easier to ma-also contribute to their specificity. Interestingly, in contrast nipulate, do not faithfully reflect important characteristicsto the requirement for dimerization to activate RTKs, di- of bona fide insulin target cells. For example, the relativemerization of PTPase domains may serve to inhibit their ca- levels of expression of IRS-1, -2, and -3 are very different intalytic activity in some cases [46]. primary adipose cells and 3T3-L1 adipocytes [25, 74, 75].

Although the determinants of PTPase specificity are not Third, the cellular machinery for appropriate subcellularcompletely understood, there is evidence that particular trafficking of GLUT4 seems to be lacking in commonlyPTPases show selectivity for specific RTKs [45]. The trans- studied cell lines such as NIH-3T3 fibroblasts, CHO cells,membrane PTPases, PTP-a, PTP-e, and LAR have all been or COS cells that do not normally express GLUT4. Thus,implicated as modulators of insulin action [47–49]. In par- even if recombinant insulin receptors and GLUT4 are over-ticular, LAR has been shown to interact with and dephos- expressed in these cells, the response to insulin with respectphorylate the insulin receptor in intact cells [50]. In addi- to translocation of GLUT4 is much less robust than in pri-tion, the expression and level of activity of LAR in insulin mary muscle or adipose cells [76].targets such as muscle and adipose tissue is increased in in- The recent use of electroporation to transfect adiposesulin resistant states such as obesity and diabetes [51, 52]. cells in primary culture in conjunction with methods forAmong the nontransmembrane PTPases, PTP1B and quantification of cell surface GLUT4 has led to a clearerSHP-2 have both been shown to modulate insulin signal- understanding of metabolic insulin signalling pathways [48,ling. PTP1B dephosphorylates the insulin receptor both in 77–86]. In addition, transgenic mice that have had key sig-vitro and in intact cells [45, 53, 54] and regulates both mito- nalling molecules either knocked out or overexpressed havegenic and metabolic actions of insulin [48, 55, 56]. In tissue provided valuable insights [35, 36, 87–90]. Finally, microin-culture models, an increase in the level and activity of jection or viral transfection strategies in differentiated 3T3-PTP1B has been associated with insulin resistance induced L1 adipocytes along with semiquantitative methods for as-by exposure to high glucose levels. In addition, the level sessing cell surface GLUT4 have been informative [91–95].and activity of PTP1B in human adipose tissue is positively Figure 3 summarizes some of what is currently known aboutcorrelated with in vivo measures of insulin sensitivity [57– insulin signalling pathways related to the translocation of60]. Binding of the SH2 domains of SHP-2 to phosphotyro- GLUT4 in adipose cells.sine motifs on either the insulin receptor or insulin receptor Many patients with syndromes of extreme insulin resis-substrate-1 (IRS-1) results in activation of SHP-2 PTPase tance have mutations resulting in inactivation of the kinaseactivity [61, 62]. Interestingly, a number of studies have domain [96]. This suggests that receptor kinase activity isshown that SHP-2 participates in Ras and MAP kinase de- necessary to mediate metabolic effects of insulin. Direct evi-pendent pathways as a positive mediator of mitogenic ac- dence that the insulin receptor tyrosine kinase is necessarytions of insulin and other growth factors [63–66]. for insulin-stimulated translocation of GLUT4 has been ob-

tained using transfected rat adipose cells in primary culture(Fig. 4) [78]. Cells overexpressing wild-type insulin recep-

INSULIN SIGNALLING PATHWAYS tors show a marked increase in cell surface GLUT4 in theRELATED TO GLUCOSE TRANSPORT

absence of insulin when compared with control cells trans-fected with an empty expression vector. In contrast, cellsA primary metabolic function of insulin that distinguishes

it from other growth factors is the promotion of whole body overexpressing a kinase-deficient mutant insulin receptorhave an insulin dose-response curve similar to that of theglucose utilization. The rate-limiting step in glucose metab-

olism under normal conditions is transport of glucose into control cells. Since the only difference between these twoexperiments is the presence of an intact kinase domain, itcells. The insulin responsive glucose transporter GLUT4 is

expressed at high levels in classic insulin targets such as can be concluded that receptor tyrosine kinase activity isnecessary for recruitment of GLUT4. Furthermore, it ismuscle and adipose tissue. Insulin stimulates increased glu-

cose transport in these tissues by causing the redistribution likely that unoccupied insulin receptors have a low level ofintrinsic tyrosine kinase activity because overexpression ofof GLUT4 from an intracellular pool to the cell surface


FIGURE 3. Insulin signalling pathways that contribute to translocation of GLUT4 in adipose cells. Interestingly, although activationof PI3K is necessary for insulin-stimulated translocation of GLUT4, it does not appear to be sufficient because activation of PI3K byPDGF is without effect on translocation of GLUT4 when PDGF receptors are expressed at physiological levels.

the wild-type receptor leads to recruitment of GLUT4 even (Fig. 5) [86]. Since this mutant is predicted to inhibit asso-ciation of all IRS proteins with the insulin receptor, thesein the absence of insulin. Also consistent with this idea are

studies showing that overexpression of PTP1B or PTPa results not only suggest that IRS family members are impor-tant mediators of metabolic actions of insulin, but also im-(PTPases that dephosphorylate the insulin receptor) signifi-

cantly decrease GLUT4 at the cell surface in the absence of ply that PI3K is an essential downstream effector. Of note,in adipose cells, the time course for the association of IRS-3insulin [48, 85, 86, 97].

Similar to overexpression of the insulin receptor, overex- with the p85 regulatory subunit of PI3K in response to insu-lin stimulation is much more rapid than for IRS-1 [98]. Inpression of insulin receptor substrates IRS-1, -2, -3, or -4 in

rat adipose cells leads to an increase in cell surface GLUT4 addition, the magnitude of the association between IRS-3and p85 in response to insulin seems to be greater than forin the absence of insulin [79, 82, 86]. Interestingly, transfec-

tion with an antisense ribozyme against IRS-1 results in a IRS-1. Furthermore, in transgenic mice lacking IRS-1,IRS-3 is the insulin receptor substrate in adipose cells re-decrease in insulin sensitivity without a decrease in maxi-

mal responsiveness with respect to translocation of GLUT4 sponsible for the majority of activation of PI3K in responseto insulin [98, 99]. Since PI3K is necessary for insulin-stim-[79]. Thus, although IRS-1 is capable of mediating the ef-

fect of insulin to stimulate translocation of GLUT4, other ulated glucose transport (see below), these data suggest thatIRS-3 may be a major insulin receptor substrate mediatingparallel pathways are probably involved. Indeed, the fact

that transgenic IRS-1 knockout mice are only mildly insu- metabolic actions in vivo.As mentioned above, two major insulin signalling path-lin resistant provides unequivocal evidence that IRS-1 con-

tributes to metabolic actions of insulin but is not absolutely ways downstream from the receptor substrates are the PI3K-dependent pathways and the Ras-dependent pathways.required for insulin-stimulated glucose uptake [35, 36]. In-

terestingly, overexpression of a mutant IRS-3 that does not Overexpression of constitutively active mutants of eitherPI3K or Ras in adipose cells leads to massive recruitment ofbind PI3K inhibits the translocation of GLUT4 in response

to insulin to a much greater extent than the IRS-1 ribozyme GLUT4 to the cell surface in the absence of insulin [80,


FIGURE 4. Insulin receptor tyrosine kinase activity is importantFIGURE 6. PI3K is necessary for insulin-stimulated translocationfor insulin-stimulated translocation of GLUT4. Rat adipose cellsof GLUT4. Rat adipose cells were co-transfected with an epitope-were co-transfected with an epitope-tagged GLUT4 and eithertagged GLUT4 and either a dominant inhibitory mutant of the p85wild-type human insulin receptors (m), tyrosine kinase-deficientregulatory subunit of PI3K (m), or an empty expression vectormutant insulin receptors cells (j), or an empty expression vector(control) (d). Cell surface concentrations of epitope-tagged(control) (d). Cell surface concentrations of epitope-taggedGLUT4 are shown as a function of insulin concentration (ex-GLUT4 are shown as a function of insulin concentration (ex-pressed as a percent of the maximally stimulated control cells) [80].pressed as a percent of the maximally stimulated control cells) [78].

100]. However, overexpression of recombinant proteins can change the response to insulin. Thus, it appears that PI3K,sometimes lead to effects that do not occur under physiolog- but not Ras, plays a necessary physiological role in theical conditions. Interestingly, when dominant inhibitory translocation of GLUT4. Interestingly, stimulation of adi-mutants are used to suppress the function of either endoge- pose cells with other growth factors such as PDGF results innous PI3K or Ras in adipose cells, overexpression of the activation of PI3K that is comparable to that seen with in-PI3K mutant results in nearly complete inhibition of insu- sulin stimulation but does not result in translocation oflin-stimulated translocation of GLUT4 (Fig. 6) [80] while GLUT4 [81, 101]. Furthermore, targeting of PI3K activityoverexpression of the Ras mutant does not significantly to GLUT4 containing vesicles is less effective at causing

translocation of GLUT4 than using non-targeted overex-pression of constitutively active PI3K in 3T3-L1 adipocytes[102]. Thus, PI3K activity per se is not sufficient to causetranslocation of GLUT4 under all conditions.

There are several effectors downstream from PI3K thatmay play a role in insulin-stimulated translocation ofGLUT4. Activation of the serine/threonine kinase Akt in-volves the lipid products of PI3K binding to the PH domainof Akt, as well as the phosphorylation of key regulatory sitesby PDK1 and PDK2 (kinases that are also activated byPI3K) [41, 103, 104]. Like PI3K and Ras, overexpression ofconstitutively active mutants of Akt in rat adipose cells or3T3-L1 adipocytes leads to massive recruitment of GLUT4to the cell surface [83, 94]. However, in contrast to the stud-ies with PI3K, overexpression of a kinase-deficient inhibi-tory mutant of Akt only partially inhibits insulin-stimulated

FIGURE 5. IRS proteins are important mediators of metabolic translocation of GLUT4 in adipose cells (Fig. 7) [83]. Thus,insulin signalling. Rat adipose cells were co-transfected with an

it is likely that multiple downstream effectors of PI3K con-epitope-tagged GLUT4 and either a mutant IRS-3 (d) unable totribute to mediating the translocation of GLUT4. For ex-bind PI3K, or an empty expression vector (control) (s) [86].

Cell surface concentrations of epitope-tagged GLUT4 are shown ample, the atypical protein kinase C isoforms PKC-z andas a function of insulin concentration (expressed as a percent of PKC-l are good candidates because they are both down-the maximally stimulated control cells). The mutant IRS-3 is pre- stream of PI3K, and insulin-stimulated translocation ofdicted to bind to the insulin receptor blocking interaction of IRS

GLUT4 and glucose transport are inhibited by overexpres-proteins with the insulin receptor and preventing IRS-mediatedactivation of PI3K in response to insulin. sion of kinase-inactive mutants of either PKC-z or PKC-l


are involved with GLUT4 trafficking. (For review see[110]). The v-SNARE VAMP2 is localized to GLUT4 con-taining vesicles in adipose cells and appears to participatein insulin-stimulated exocytosis of GLUT4 [111–113]. Thet-SNARE Syntaxin 4 binds specifically to VAMP2 and islocalized to the plasma membrane in muscle and adiposecells. Insulin-stimulated translocation of GLUT4 in 3T3-L1adipocytes can be blocked by using antibodies against Syn-taxin 4 or overexpressing the cytoplasmic tail of Syntaxin4 [113–115]. Although a major effect of insulin is to in-crease the exocytosis rate for GLUT4, endocytosis is also animportant determinant of cell surface transporter levelssince overexpression of a dominant inhibitory mutant of dy-namin (a GTPase necessary for endocytosis) results in accu-mulation of GLUT4 in the plasma membrane in adipocytes[116, 117]. An important goal of current investigations re-lated to the metabolic actions of insulin is to understandhow insulin signalling is linked to trafficking of GLUT4.

MECHANISMS FOR ACHIEVING SPECIFICITY

Although multiple downstream effectors of insulin actionare shared in common by many RTKs, there are potentialmechanisms for incorporating specificity at each step in theinsulin signal transduction pathway.

Specificity at the Receptor Level

The binding affinity between insulin and its receptor isquite high and provides an obvious first determinant of sig-nal specificity. However, insulin is also capable of bindingand activating other related receptors such as the IGF-1 re-

FIGURE 7. Physiological role for Akt in insulin-stimulated ceptor. Similarly, IGF-1 can bind to and activate the insulintranslocation of GLUT4. Rat adipose cells were co-transfected

receptor [118]. The binding affinities of insulin and IGF-1with an epitope-tagged GLUT4 and either (A) wild-type Aktfor their heterologous receptors are approximately 100-fold(d), (B) a kinase inactive mutant Akt (m), or (A, B)an emptyless than for their own receptors. If receptor occupancy isexpression vector (control) (s). Cell surface concentrations of

epitope-tagged GLUT4 are shown as a function of insulin con- proportional to signal amplitude, this differential bindingcentration (expressed as a percent of the maximally stimulated affinity may lead to the integration of multiple signals atcontrol cells) [83]. varying amplitudes and contribute to the determination of

specific effects. Furthermore, since the receptors for insulinand IGF-1 share substantial homology, formation of hybrid

in adipocytes [105–107]. Interestingly, it was recently receptors consisting of one ab subunit of the insulin recep-shown that PDK1, in addition to contributing to activation tor and one IGF-1 ab subunit is possible and the signalsof Akt, binds and phosphorylates PKC-z [108]. generated by these hybrids may be unique [119,120]. In ad-

A complementary approach to tracing metabolic path- dition, the relative level of expression of insulin and IGF-Iways originating from the insulin receptor has been to study receptors is tissue-specific so that the number of hybrid re-the trafficking mechanisms for GLUT4 and to investigate ceptors varies in each tissue. For example, in vascular endo-how these processes are linked to upstream insulin signal- thelial cells that normally express 10 times as many IGF-1ling pathways. Interestingly, mechanisms that determine receptors as insulin receptors, stimulation with insulin atvesicular trafficking during regulated exocytosis of synaptic concentrations sufficient to saturate both IGF-1 and insulinvesicles in neurons also apply to the movement of GLUT4. receptors results in the production of nitric oxide at a levelIn general, vesicle docking and fusion to the plasma mem- that is twice that seen with stimulation by IGF-1 at compa-brane is mediated by specific interactions of soluble rable concentrations [121].N-ethylmaleimide-sensitive factor attachment protein re- Another feature of the insulin receptor that may affectceptors (SNAREs) [109]. v-SNARE proteins are localized signal specificity is negative cooperative binding interac-to the vesicle membrane while t-SNARE proteins are local- tions with insulin [122]. That is, the binding affinity of insu-ized to the target plasma membrane. There is good evidence lin for its receptor decreases with increasing insulin concen-

trations. Thus, the dynamics of intracellular signallingthat specific isoforms of v-SNARE and t-SNARE molecules


Compartmentalization of Signalling Complexesevents in response to a particular insulin secretory profilemay encode some specificity. Additional evidence that the Signal specificity may also be determined, in part, by local-binding interaction between ligand and receptor affects sig- ization of signalling complexes to particular subcellularnalling specificity comes from studies with point mutants of compartments. For example, in adipocytes, stimulation byinsulin molecules that have been designed to have higher either insulin or PDGF results in comparable increases inbinding affinities for the insulin receptor than the native whole cell PI3K activity, yet only the PI3K activity stimu-insulin molecule. For example, the Asp B10 insulin mutant lated by insulin causes translocation of GLUT4 [81, 101].has a much higher binding affinity than native insulin for Interestingly, insulin stimulation localizes IRS-1/PI3K com-the insulin receptor and appears to favor mitogenic rather plexes to GLUT4 containing vesicles in adipose cells [133].than metabolic actions of insulin [123]. However, when PI3K activity is experimentally targeted to

Finally, integration of signals generated by cross-talk be- the GLUT4 containing vesicles, this is not sufficient totween different types of receptors may contribute to the cause translocation of GLUT4 or increases in glucose trans-specificity of insulin action. For example, in addition to the port that are comparable to that seen with insulin stimula-well known cross-talk that occurs between insulin and tion [102]. In addition, when PDGF receptors are overex-IGF-1 at the receptor level, there is evidence for cross-talk pressed in adipose cells, stimulation with PDGF results inbetween insulin and PDGF signalling with respect to inter- translocation of GLUT4 that is comparable to that seenactions of IRS-1 and PI3K [124]. Another example of cross- with insulin stimulation. These studies are consistent withtalk involves G-protein coupled receptors such as the angio- the possibility that overexpression of the PDGF receptortensin II receptor that can interact with IRS-1 and result in leads to aberrant localization of the receptor and other sig-modulation of insulin signalling [125–127]. Furthermore, nalling molecules into compartments from which theythe insulin receptor is capable of phosphorylating the would normally be excluded [81]. Thus, although localiza-G-protein coupled b-adrenergic receptor causing attenua- tion of PI3K activity appears to be important for metaboliction of cyclic AMP production in response to b-adrenergic actions of insulin, the precise subcellular compartment thatagonists (for review see [128]). Signal cross-talk may also is related to translocation of GLUT4 has yet to be deter-mediate specificity that is determined by the external envi- mined.ronment of the cells. For example, stimulation of a5b1-inte- Another example of differential subcellular compartmen-grins by fibronectin enhances insulin receptor and IRS-1 talization leading to potential signal specificity is the intra-phosphorylation [129]. Specific members of the integrin cellular distribution of IRS-1, -2, and -3. Upon subcellularfamily bind to particular extracellular matrix proteins. fractionation of 3T3-L1 adipoctyes, IRS-1 and -2 are foundThus, the extracellular matrix surrounding the cell may in- mainly in the low density microsome fraction while IRS-3fluence the specificity of signalling by the insulin receptor. is found predominantly in the plasma membrane fraction

[134]. It should be noted that IRS proteins are probably as-sociated with cytoskeletal elements that co-purify withSpecificity at the Receptor Substrate Levelthese membrane fractions rather than with the membranesThe existence of multiple substrates of the insulin receptorthemselves [135]. Nevertheless, this differential localizationprovides additional opportunities to incorporate specificity.of IRS isoforms may contribute to targeting downstream ef-Members of the IRS family of substrates contain numerousfectors such as PI3K to specific subcellular compartments.phosphotyrosine docking sites for SH2-domain containingIndeed, qualitatively different effects on translocation ofproteins. The number of these docking sites and the partic-GLUT4 in adipose cells are observed when overexpressionular SH2 domains with which they interact vary among theof IRS-1 is compared with overexpression of IRS-3 [86]. Indifferent IRS proteins. That is, the combination of down-addition to specific intracellular compartments, the forma-stream signalling molecules engaged by an IRS protein, astion of signalling microdomains is another potential mecha-well as the relative affinities of particular downstream ef-nism for segregating molecules that may contribute to speci-fectors for each substrate, are unique for each IRS protein.ficity. For example, adipose cells (and many other highlyThus, tissue-specific differences in the relative expressiondifferentiated cells) are known to contain large numbers oflevels of these IRS substrates may result in formation of dis-caveolae in their plasma membrane. Caveolae are smalltinct signalling complexes in particular tissues and help toflask-shaped invaginations in the plasma membrane thatexplain why some actions of insulin predominate in certaincontain ceramide, glycosphingolipids, cholesterol, and scaf-tissues [74, 130]. Some downstream signalling moleculesfold-like proteins known as caveolin [136, 137]. Overex-contain tandem SH2 domains (e.g., SHP-2 and the p85 reg-pression of caveolin is sufficient to mediate the formationulatory subunit of PI3K) so that the spatial relationship be-of caveolae [138]. The insulin receptor and many other re-tween these SH2 domains provides an additional level ofceptor tyrosine kinases (e.g., PDGF and EGF receptors)specificity. That is, the geometry of multiple phosphotyro-have particular aromatic amino acid motifs in their kinasesine motifs on a particular substrate is important for optimaldomain (FXFXXXXF) that bind to the so-called scaffold-binding and activation of proteins with tandem SH2 do-ing domain of caveolin in vitro and presumably localizemains [131, 132]. Similarly, the relationship of SH2 andthese receptors to caveolae [139–142]. Recently, in humanSH3 domains in various adaptor or effector molecules mayfibroblasts, it was shown that the PDGF receptor, Ras, Raf1,impose physical constraints on the formation of signalling

complexes that are important for signal specificity. MEK1, and ERK2 (MAP kinase isoform) are all localized to


caveolae in the absence of PDGF. Upon PDGF stimulation mains [154]. Thus, the combination of different isoforms ofregulatory and catalytic subunits of PI3K in conjunctionof the intact cells, phosphorylation of ERK2 is increased in

the caveolar fraction. Furthermore, in vitro stimulation of with tissue specific expression and localization to subcellu-lar compartments may result in the generation of a particu-the isolated caveolar fraction with PDGF also results in

phosphorylation of ERK2. Thus, all of the signalling com- lar profile of lipid products that interact in specific wayswith downstream effectors that determines the biologicalponents necessary for PDGF-stimulated MAP kinase acti-

vation are localized to functional microdomains created by response to insulin.caveolae [141]. Interestingly, the number of caveolae, andthe expression of caveolin, increases substantially upon dif- Feedback Regulationferentiation of 3T3-L1 fibroblasts into adipocytes [143,

The function of end-products to dampen or amplify signals144]. Although the fraction of particular RTKs such as thefrom upstream pathways is a common paradigm used for theinsulin receptor that are associated with caveolae is likelyregulation of enzymes. It is possible that specificity in RTKto be relatively small, it is conceivable that caveolae and ca-signal transduction is also determined, in part, by positiveveolin contribute to the specificity of metabolic insulin sig-or negative feedback. In the case of insulin signalling, it wasnalling pathways in adipose cells.recently shown that GSK-3 (a serine/threonine kinasedownstream from Akt) can phosphorylate IRS-1 and in-

Tissue-Specific Expression of Key Effectors hibit insulin receptor tyrosine kinase activity [155]. Simi-Signal specificity may also be determined by tissue-specific larly, PI3K has serine/threonine kinase activity in additionexpression of crucial molecules. For example, GLUT4 is to its lipid kinase activity that can phosphorylate IRS-1 andpredominantly expressed in classical insulin targets such as may modulate IRS-1 function [156]. In addition, there is ev-muscle and adipose tissue. Other tissues are not nearly as re- idence that PI3K has functional interactions both upstreamsponsive to insulin with respect to glucose transport, in part and downstream from Ras suggesting another feedback loopbecause they do not express GLUT4 or high levels of insu- that may be involved with insulin signalling [157, 158].lin receptors. Furthermore, there are presumably additionaltissue-specific signalling elements necessary for insulin- Modulation of Signal Frequency and Amplitudestimulated glucose transport because expression of recombi-

Cellular signals generated by changes in calcium fluxes ornant insulin receptors and GLUT4 in cells derived from in-membrane potential often encode information in the mod-sulin-insensitive tissues is not sufficient to enable a responseulation of signal frequency and amplitude. Although regula-to insulin comparable to that observed in bona fide insulintion of intracellular calcium dynamics is not a well appreci-target cells. Another finding consistent with the idea thatated feature of insulin signalling, many RTKs including thetissue-specific expression is important is that phosphoryla-insulin receptor can mediate increases in intracellular cal-tion of caveolin in response to insulin stimulation occurs incium [159]. Recent data link PI3K activation to calciumdifferentiated 3T3-L1 adipocytes but not in undifferentiatedregulation via PLC-g [160] Lipid products of PI3K such as3T3-L1 fibroblasts [145]. The insulin signalling pathway re-phosphatidylinositol (3,4,5,) tri-phosphate (PI(3,4,5,)P3)lated to caveolin phosphorylation includes phosphorylatedcan bind to the PH domain of PLC-g resulting in its activa-c-cbl activating fyn which then directly phosphorylates caveo-tion. In addition, PI(3,4,5,)P3 can also bind to the PH do-lin. Interestingly, although the insulin receptor, c-cbl, fyn,main of TEC family kinases resulting in their activation andand caveolin are all expressed in both 3T3-L1 adipocytessubsequent tyrosine phosphorylation and activation ofand fibroblasts, insulin stimulation results in phosphoryla-PLC-g [161]. Activated PLC-g increases the levels of inosi-tion of c-cbl only in the differentiated 3T3-L1 adipocytetol tri-phosphate (IP3) leading to the release of intracellular[145, 146]. This implies that the kinase responsible forcalcium. Further evidence that PI3K can regulate intracel-phosphorylation of c-cbl (or some other upstream compo-lular calcium levels comes from the observation that inhibi-nent) is expressed only in the adipocyte but not the fibro-tion of PI3K can block PLC-g mediated IP3 productionblast form of 3T3-L1 cells and may explain why caveolin is[161]. Thus, a potential mechanism for insulin to controlphosphorylated in response to insulin only in adipocytes.the dynamics of intracellular calcium exists. It is also con-The existence of multiple isoforms of key signalling mol-

ecules may also be important for signal specificity. For ex- ceivable that the dynamics of signalling by RTKs encodesspecific information by modulating the frequency and am-ample, PI3K is essential for insulin-stimulated glucose trans-

port. However, multiple isoforms and splice variants of both plitude of various phosphorylation cascades. As pointed outbefore, the time course for association between PI3K andthe regulatory p85 subunit and catalytic p110 subunit of

PI3K that have differential responses to insulin have been IRS-3 in rat adipose cells in response to insulin stimulationis faster than for IRS-1 [98]. Furthermore, in the same study,discovered [147–153]. Each of these isoforms may generate

a distinct pattern of lipid products that have specific roles the amount of PI3K associated with IRS-3 in response to in-sulin stimulation was also greater than for IRS-1. This dif-in signalling. The lipid products of PI3K are known to bind

to PH domains of downstream effectors resulting in activa- ference in the time course and amplitude of PI3K activationmay help to distinguish signals that are mediated by IRS-1tion or regulation of these PH domain-containing mole-

cules. Recently, various lipid products of PI3K were shown from those mediated by IRS-3 and ultimately result in dif-ferent biological effects.to have differential binding affinities for particular PH do-


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