Identification of the rat adapter Grb14 as an inhibitor of insulin actions.
We cloned by interaction with the beta-subunit of the insulin receptor the rat variant of the human adapter Grb14 (rGrb14). rGrb14 is specifically expressed in rat insulin-sensitive tissues and in the brain. The binding of rGrb14 to insulin receptors is insulin-dependent in vivo in Chinese hamster ovary (CHO) cells overexpressing both proteins and importantly, in rat liver expressing physiological levels of proteins. However, rGrb14 is not a substrate of the tyrosine kinase of the receptor. In the two-hybrid system, two domains of rGrb14 can mediate the interaction with insulin receptors: the Src homology 2 (SH2) domain and a region between the PH and SH2 domains that we named PIR (for phosphorylated insulin receptor-interacting region). In vitro interaction assays using deletion mutants of rGrb14 show that the PIR, but not the SH2 domain, is able to coprecipitate insulin receptors, suggesting that the PIR is the major binding domain of rGrb14. The interaction between rGrb14 and the insulin receptors is almost abolished by mutating tyrosine residue Tyr1150 or Tyr1151 of the receptor. The overexpression of rGrb14 in CHO-IR cells decreases insulin stimulation of both DNA and glycogen synthesis. These effects are accompanied by a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1, but insulin receptor autophosphorylation is unaltered. These findings suggest that rGrb14 could be a new downstream signaling component of the insulin-mediated pathways.
Kasus-Jacobi
A
A
Endocrinologie Métabolisme et Developpement, CNRS, UPR 1524, 9 rue Jules Hetzel, 92190 Meudon, France.
Perdereau
D
D
Auzan
C
C
Clauser
E
E
Van Obberghen
E
E
Mauvais-Jarvis
F
F
Girard
J
J
Burnol
A F
AF
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D007328:Insulin / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011506:Proteins / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D015398:Signal Transduction / Q000502:physiology*; D018909:src Homology Domains / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; C490976:Grb14 protein, rat; D007328:Insulin; D011506:Proteins; D012333:RNA, Messenger; D019000:Phosphotyrosine; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin