Abstract
cDNA clones encoding human (h) Grb7 and a previously unknown protein with high homology to hGrb-IR and mGrb10 (where m indicates mouse) were found by screening expressed sequence tag data bases. hGrb7 mRNA expression is greatest in pancreas and restricted to a few other tissues. The second protein termed hGrb-IRbeta/Grb10 contains an intact PH domain and lacks the 80-residue mGrb10 insertion. Expression is greatest in pancreas and muscle but occurs in nearly all tissues. hGrb-IRbeta/Grb10 and hGrb-IR likely arise as alternative mRNA splicing products of a common gene. Reverse transcriptase-coupled polymerase chain reaction shows both mRNAs in muscle. In cells, Grb-IRbeta/Grb10 protein translocates from cytosol to membrane upon insulin stimulation, most likely due to direct interactions with the insulin receptor. These interactions are mediated by the SH2 domain and additional regions of the protein. Studies with mutated receptors and synthetic phosphopeptides show that the hGrb-IRbeta/Grb10 SH2 domain binds at least two sites in the insulin receptor: the kinase activation loop > the juxtamembrane site. hGrb-IRbeta/Grb10 also binds a 135-kDa phosphoprotein in unstimulated 3T3-L1 adipocytes; binding is reduced upon insulin stimulation. In addition, the c-Abl SH3 domain binds Grb-IR/Grb10, whereas Fyn, phosphatidylinositol 3-kinase p85, and Grb2 SH3 domains do not. The site of c-Abl SH3 domain interaction is highly conserved within the Grb-IR/Grb10/Grb7/Grb14 family. hGrb-IRbeta/Grb10 also binds platelet-derived growth factor and epidermal growth factor receptors, suggesting a broader role in the signaling pathways of numerous receptors. We conclude that hGrb-IRbeta/Grb10 is a widely expressed, PH and SH2 domain-containing, SH3 domain-binding protein that functions downstream from activated insulin and growth factor receptors.
Highlights
Many of the effects of activated tyrosine kinase-linked receptors are mediated by cascades of intracellular tyrosine phosphorylation reactions
Homology to hGrb-IR and mGrb10, which we refer to as hGrbIR/Grb10. It has an intact PH domain and lacks the mGrb10 insertion. hGrb-IR/Grb10 and hGrb-IR probably represent alternative mRNA splicing products of a common gene. Both mRNAs are expressed in muscle, a major site of insulin action. hGrb-IR/Grb10 protein is present in the cytosol of unstimulated Rat1 fibroblasts and translocates to the membrane following insulin stimulation
Expressed sequence tags typically represent incomplete gene sequences, partial protein coding sequences can be deduced from the data, and in some cases it is possible to predict the function of the encoded protein based on sequence homology
Summary
Many of the effects of activated tyrosine kinase-linked receptors are mediated by cascades of intracellular tyrosine phosphorylation reactions. In the case of insulin signaling, autophosphorylation activates the receptor kinase [3, 4] and creates a docking site for substrate protein PTB domains [5, 6]. Homology to hGrb-IR and mGrb, which we refer to as hGrbIR/Grb10 It has an intact PH domain and lacks the mGrb insertion. HGrb-IR/Grb and hGrb-IR probably represent alternative mRNA splicing products of a common gene Both mRNAs are expressed in muscle, a major site of insulin action. We have characterized hGrb-IR/ Grb interactions with activated insulin, EGF, and PDGF receptors and SH3 domain proteins. We conclude that Grb-IR/ Grb is a previously unknown signaling protein that may function downstream from activated insulin and growth factor receptors
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