IRSProtein Structure and Function

Alignment of the amino acid sequences of the IRS-proteins reveals important similarities and subtle differences (Fig. 1). IRS 1-4 and Chico contain an NH2-terminal pleckstrin homology (PH) domain adjacent to a PTB domain. The structures of the PH and PTB domains are remarkably similar [30], and both facilitate recruitment of IRS-proteins to the activated insulin and IGF1 receptors; deletion of the PH and PTB domain almost completely prevents phosphoryla-tion of the C-terminus. The PTB domain binds to the phos-phorylated NPEY motif in the P-subunit of the insulin or IGF1 receptor [31-33]. At ordinary expression levels, deletion of the PTB domain reduces that ability of insulin to promote tyrosine phosphorylation of IRS1 or IRS2; however, overexpression of the insulin receptor restores phosphoryla-tion and signaling, suggesting that the PTB domain enhances coupling but is not essential for signaling [31].

The PH domain also promotes interaction between IRS-proteins and physiological levels of insulin receptors. Like the PTB domain, the PH domain is not required in cells overexpressing the insulin receptor [31]. However, the mechanism of coupling employed by the PH domain is not understood. Some, but certainly not all, PH domains bind to membrane phospholipids which provides membrane targeting [34]; however, the PH domain in IRS-proteins has a relatively low affinity for phospholipids. The PH domains can be exchanged between IRS-proteins without noticeable loss of bioactivity, but chimeric IRS-proteins composed of het-erologous PH domains fail to be phosphorylated by the insulin receptor [35]. Because IRS-protein PH domains do not bind to the insulin receptor, other proteins of membrane lipids might be involved. Yeast two-hybrid screens reveal a few potential binding partners, including nucleolin or a novel protein called PHIP [35,36].

The tyrosine phosphorylation sites in the COOH-terminal end of each IRS-protein recruit and regulate various downstream signaling proteins (Fig. 1). IRS1 and IRS2 have the longest tails, which contain 20 potential tyrosine phospho-rylation sites; however, only a few sites have been formally identified, and little information is available on the phos-phorylation sites in IRS3 and IRS4 (Fig. 1). Many of the tyrosine residues cluster into common motifs that recruit or activate enzymes (PI 3-kinase, SHP2, fyn) or adapter molecules (Grb-2, nck, crk, Sh2b) (Fig. 1). Grb2 and possibly SHP2 couple Grb2/SOS to IRS-proteins, which promotes the ras ^ raf cascade [37]. All IRS-proteins contain multiple p85 binding motifs that recruit PI 3-kinase, which is the best-studied insulin-signaling pathway.

In addition to the tyrosine phosphorylation sites, sequence alignment of IRS-proteins reveals several conserved motifs that might be binding sites for other cellular proteins. One of the best characterized is the binding site for the c-Jun N-terminal kinase (JNK), which resembles the sites in the JNK-interacting proteins (JIP1 and JIP2) [38,39]. This site occurs in IRS1, IRS2, and Chico but has only been validated in mouse and human IRS1 [40]. During stimulation by proin-flammatory cytokines or by insulin, JNK binds to IRS1 and promotes phosphorylation of Ser312 in human IRS1 (Ser307 in rat/mouse IRS1) [41]. Phosphorylation of this serine residue inhibits binding of the PTB domain to the phospho-rylated NPEY motif in the activated insulin receptor [40]. JNK also phosphorylates IRS2, but the homologous site is absent and the inhibitory mechanism is poorly described.

IRS1 and IRS2 contain several putative 14-3-3 binding sites (Fig. 1). 14-3-3 proteins are highly conserved acidic scaffold proteins that bind to specific amino acid sequence motifs. In some cases, serine phosphorylation of the target motif directs the binding of a 14-3-3 isoform, which might inhibit signaling by sequestering the complex to an inappropriate subcellular compartments [42]. IRS1 and IRS2 contain several serine phosphorylation motifs that bind to 14-3-3e or 14-3-3Z might inhibit signaling [43,44]. Some of these sites are in the PTB domain, which might inhibit coupling to the activated insulin receptor. Recent evidence suggests that 14-3-3 binding might displace serine-phosphorylated IRS-1 from particular structures, thus reducing PI 3-kinase activity [45]; however, a role for 14-3-3 binding in IRS-protein regulation remains to be validated in animal models. Whether 14-3-3 binding regulates IRS2 function is unknown.

Work with transgenic mice reveals that IRS1 and IRS2 have distinct signaling potential even though the structures of the two proteins and their functions in cell-based assays are rather similar; however, certain differences do exist that might establish specificity. For example, IRS2 contains a unique region of undefined structure that binds to the phos-phorylated regulatory loop of the insulin receptor kinase, called the kinase-regulatory-loop-binding (KRLB) domain. The discovery of this interaction was unexpected, as it maps to the portion of the COOH-terminal region between amino acid residues 591 and 786 that contains tyrosine phosphorylation sites (Fig. 1). Two tyrosine residues in the KRLB domain at positions 628 and 632 are crucial for this interaction. Phosphorylation of tyrosine residues in the KRLB domain by the insulin receptor inhibits the binding to the receptor, suggesting that a novel mechanism regulates the interaction of the insulin receptor and IRS-2 and may be able to distinguish the signal of IRS2 from IRS1 [46].

Figure 1 IRS-protein structures; a comparison of important sequence features of human IRS1, IRS2, and IRS4, mouse IRS4, and Drosophila Chico. The relative positions of the pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are indicated. The relative positions of potential tyrosine phosphorylation sites are also shown, as are other known interaction motifs, including sites that bind the N-terminal c-Jun kinase (JNK) 14-3-3 proteins. Alanine- and proline-rich motifs are also highlighted.

Figure 1 IRS-protein structures; a comparison of important sequence features of human IRS1, IRS2, and IRS4, mouse IRS4, and Drosophila Chico. The relative positions of the pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are indicated. The relative positions of potential tyrosine phosphorylation sites are also shown, as are other known interaction motifs, including sites that bind the N-terminal c-Jun kinase (JNK) 14-3-3 proteins. Alanine- and proline-rich motifs are also highlighted.

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