Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I-induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I-dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I-stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I-induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I-stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I-dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the ␤3 subunit of the ␣V␤3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I-dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I-stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-Idependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs. INTRODUCTIONBoth vascular smooth muscle cell proliferation and migration in response to growth factor stimulation play important roles in the formation of atherosclerotic plaques, and insulin-like growth factor I (IGF-I) is a potent stimulator of smooth muscle cell proliferation and migration (Jones et al., 1996). In primary cultured smooth muscle cells (pSMCs), IGF-I induces activation of both the phosphatidylinositol (PI)-3 kinase and the mitogen-activated protein (MAP) kinase (MAPK) pathways. Both pathways have been shown to play roles in mediating IGF-I-dependent cell migration and cell proliferation responses (Imai and Clemmons, 1999;Maile et al., 2003).Previous studies have shown that the protein tyrosine phosphatase SHP-2 plays an impor...

Insulin-like growth factor I (IGF-I) is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the devel

This study examines circulating and follicular hormone concentrations and fertilization of oocytes in cycles totally unperturbed by exogenous gonadotrophins in 10 women (25 cycles) with untreated minimal-mild endometriosis and nine women (23 cycles) with prolonged unexplained infertility compared with 16 women (50 cycles) with tubal damage as functional controls. Endometriosis was associated with a significantly longer follicular phase (median 15, 12, 13 days respectively) and reduced oestrogen secretion (median index area under the curve 3063, 3842, 3805 units respectively) compared with controls. Both endometriosis and unexplained infertility had significantly reduced serum luteinizing hormone (LH) surges [median peak serum (LH) 43, 39, 55 IU/l respectively and median area under the curve 661, 687, 823 units respectively] and reduced LH concentrations in follicular fluid (median 19.6, 10.6, 9.2 IU/l respectively). These findings suggest that infertility associated with minor endometriosis and of apparently unexplained aetiology share a common pathophysiology in impaired LH surge secretion. Whether that represents a primary pituitary disorder or is secondary to a defective ovarian signal is discussed.

Activation of insulin-like growth factor I receptor (IGF-IR) kinase is an important site of control of IGF-Ilinked intracellular signaling pathways. One potentially important regulatory variable is IGF-IR dephosphorylation. It has been shown that SHP-2, a tyrosine phosphatase, can bind to the activated IGF-IR in vitro; however, its role in IGF-IR dephosphorylation in whole cells is unknown. These studies were undertaken to determine whether SHP-2 was a candidate for mediating IGF-IR dephosphorylation. The IGF-IR in smooth muscle cells was dephosphorylated rapidly beginning 10 min after ligand addition, and this was temporally associated with SHP-2 binding to the receptor. IGF-I stimulated SHPS-1 phosphorylation and the subsequent recruitment of SHP-2. In cells expressing a SHPS-1 mutant that did not bind SHP-2 there was no recruitment of SHP-2 to the IGF-IR. Cells expressing a catalytically inactive form of SHP-2 showed SHP-2 recruitment to SHPS-1, but this did not result in SHPS-1 dephosphorylation, and there was a prolonged IGF-IR phosphorylation response after IGF-I stimulation. These studies indicate that IGF-IR stimulates phosphorylation of SHPS-1 which is critical for SHP-2 recruitment to the plasma membrane and for its recruitment to the IGF-IR. Recruitment of SHP-2 to the receptor then results in receptor dephosphorylation. The regulation of this process may be an important determinant of IGF-IR-mediated signaling.The insulin-like growth factor I (IGF-I) 1 receptor (IGF-IR) is composed of two extracellular subunits and two transmembrane ␤ subunits. Upon ligand binding, the kinase domain of the receptor autophosphorylates several tyrosine residues that are located in the intracellular domains of the ␤ subunits (1-3). This provides binding sites for other molecules, including insulin receptor substrate 1 (IRS-1) and SHC (4, 5), and the activated IGF-IR transmits downstream signals via tyrosine phosphorylation of these molecules. Activation of these tyrosines is not only critical for the downstream signaling, but also for sustained stimulation of IGF-I-stimulated biologic responses. Mutation of specific tyrosines in the receptor has been shown to result in attenuation of the ability of IGF-I to stimulate DNA synthesis, cell migration, and inhibition of apoptosis (6). Most important is the triple tyrosine motif that is phosphorylated rapidly in response to catalytic activation of the receptor, and mutation of any of these residues results in loss of signaling and IGF-I responsiveness (7).The tyrosine phosphorylation status of proteins in growth factor signaling pathways is maintained by a balance between the activity of tyrosine kinases and tyrosine phosphatases. Because phosphorylation of these tyrosines in the receptor ␤ subunit is so important for IGF-I signaling it has been assumed that dephosphorylation of the receptor would result in appropriate regulatory control of this signaling stimulus. The time course of phosphorylation of tyrosine kinase-containing growth factor receptors has been shown to be bi...