The protein IRS-1 acts as an interface between signalling proteins with Src-homology-2 domains (SH2 proteins) and the receptors for insulin, IGF-1, growth hormone, several interleukins (IL-4, IL-9, IL-13) and other cytokines. It regulates gene expression and stimulates mitogenesis, and appears to mediate insulin/IGF-1-stimulated glucose transport. Thus, survival of the IRS-1-/- mouse with only mild resistance to insulin was surprising. This dilemma is provisionally resolved with our discovery of a second IRS-signalling protein. We purified and cloned a likely candidate called 4PS from myeloid progenitor cells and, because of its resemblance to IRS-1, we designate it IRS-2. Alignment of the sequences of IRS-2 and IRS-1 revealed a highly conserved amino terminus containing a pleckstrin-homology domain and a phosphotyrosine-binding domain, and a poorly conserved carboxy terminus containing several tyrosine phosphorylation motifs. IRS-2 is expressed in many cells, including tissues from IRS-1-/- mice, and may be essential for signalling by several receptor systems.

Although several interleukin-3 (IL-3)-dependent cell lines proliferate in response to IL-4 or insulin, the 32D line does not. Insulin and IL-4 sensitivity was restored to 32D cells by expression of IRS-1, the principal substrate of the insulin receptor. Although 32D cells possessed receptors for both factors, they lacked the IRS-1--related protein, 4PS, which becomes phosphorylated by tyrosine in insulin- or IL-4--responsive lines after stimulation. These results indicate that factors that bind unrelated receptors can use similar mitogenic signaling pathways in hematopoietic cells and that 4PS and IRS-1 are functionally similar proteins that are essential for insulin- and IL-4--induced proliferation.

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D. E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3-EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D. E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.

; IRS-1 YCT was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-1 3YMXM nor IRS-1 YCT mediated activation of mitogen-activated protein kinases. IRS-1 F18 and IRS-1 YCT partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phosphotyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-1 YCT do not augment this signal. IRS-1 3YMXM mediated the maximal mitogenic response to insulin, although the response to insulin was more sensitive with wild-type IRS-1. By contrast, the association of IRS-1 3YMXM with PI 3-kinase was more sensitive to insulin than the association with IRS-1. Thus, the binding of SH2 proteins (such as PI 3-kinase) by YMXM motifs in IRS-1 is an important element in the mitogenic response, but other elements are essential for full mitogenic sensitivity.The IRS proteins (IRS-1 and IRS-2) are endogenous cellular substrates of the insulin and insulin-like growth factor 1 (IGF-1) receptor tyrosine kinases, as well as substrates for various cytokine receptors which activate various members of the Janus kinase family (22,41,42,47,48). Tyrosine phosphorylation plays a central role in the mechanism of IRS protein signaling by providing docking sites for proteins containing isoforms of the Src homology 2 (SH2) domain (25,38,39). Since the phosphorylation sites in IRS-1 are situated in various amino acid sequence motifs, IRS-1 engages numerous SH2 proteins, including p85, Grb-2, Fyn, Nck, and SHP2 (SHPTP2, Syp, and PTP1D) (10,15,36). IRS proteins contain several phosphorylation sites in YXXM motifs, which are selective binding sites for the SH2 domains in the regulatory subunits (p85 and p55 PIK ) of phosphatidylinositol (PI) 3Ј-kinase (22,28,41). During insulin stimulation, IRS proteins mediate the direct activation of PI 3Ј-kinase, which appears to play essential roles in the activation of the p70 s6k and the akt kinase (21, 22) and the serine phosphorylation of eIF4e and PHAS-I as well as in mediating various biological responses including vesicle movement (including glucose transporter translocation), mitogenesis, general protein synthesis, receptor endocytosis, and chemotaxis, among other things (22,25,48).While the plethora of signaling events apparently mediated by PI 3Ј-kinase raises questions about whether PI 3Ј-kinase is a regulator of specific cellular events or whether it is a global potentiator of cellular signaling, it is obviously an important molecule in the transmission of downstream signals by tyrosine kinases. The 110-kDa PI 3Ј-kinases are coupled to tyrosine kinases by regulatory adapter molecules (p85 and p55 PIK ) which use their SH2 domains to bind tyrosyl-phosphorylated YMXM motifs (14, 28). The 110-kDa catalytic domain (p110) is activated by the binding of these tyrosyl-phosphorylated YMXM motifs (2). In addition to its ability to phosphorylate PI, p110 also phosphorylates certain proteins on serine residues (3,8,14,17). The SH2 d...