CLONING, CHROMOSOME LOCALIZATION, EXPRESSION, AND CHARACTERIZATION OFAN SRC HOMOLOGY-2 AND PLECKSTRIN HOMOLOGY DOMAIN-CONTAINING INSULIN-RECEPTOR BINDING-PROTEIN HGRB10-GAMMA

hGrb10 alpha (previously named Grb-IR) is a Src-homology 2 domain-cont aining protein that binds with high affinity to the tyrosine-phosphory lated insulin receptor and insulin-like growth factor-1 receptor, At l east two isoforms of human Grb10, (hGrb10 alpha and hGrb10 beta), whic h differ in the pleckstrin homology (PEI) domain and the N-terminal se quence, have previously been identified in insulin target tissues such as human skeletal muscle and fat cells, Here we report the cloning of the third isoform of the hGrb10 family (hGrb10 gamma) from human skel etal muscle and its localization to human chromosome 7, We have also d etermined the human chromosome localization of Grb7 to 17q21-q22 and G rb14 to chromosome 2, hGrb10 gamma contains an intact PEI domain and a n N-terminal sequence that is present in hGrb10 gamma but absent in hG rb10 beta, RNase protection assays and Western blot analysis showed th at hGrb10 alpha and hGrb10 gamma are differentially expressed in insul in target cells including skeletal muscle, liver, and adipocyte cells, hGrb10 gamma is also expressed in HeLa cells and various breast cance r cell lines, The protein bound with high affinity to the insulin rece ptor in cells, and the interaction was dependent on the tyrosine phosp horylation of the receptor, hGrb10 gamma also underwent insulin-stimul ated membrane translocation and serine phosphorylation, hGrb10 gamma p hosphorylation was inhibited by PD98059, a specific inhibitor of mitog en-activated protein kinase kinase, and wortmannin, a specific inhibit or of phosphatidylinositol 3-kinase, Taken together, our data suggest that hGrb10 isoforms are potential downstream signaling components of the insulin receptor tyrosine kinase and that the PH domain may play a n important role in the involvement of these isoforms in signal transd uction pathways initiated by insulin and other growth factors.