Demonstration of putative subunit(s) of a novel insulin receptor-associated 66 kDa GTP-binding protein, Gir.
A 66 kDa GTP-binding protein, Gir, and insulin receptor (IR) were copurified from human placental membrane by DEAE-Sephacel and Wheat Germ Agglutinin (WGA)-Sepharose affinity chromatography. The WGA-fraction containing IR and Gir (IR-Gir-fraction) was phosphorylated (95 kDa IR-beta and 66 kDa Gir) by IR-tyrosine kinase using [32P]ATP or photolabeled with [32P]8-azido-GTP (mainly 66 kDa), and was cross-linked with a bifunctional reagent, bis-[sulfosuccinimidyl] suberate (BS3). The Gir cross-linked with putative subunit(s) to form a 110 kDa complex. Phosphorylation as well as 8-azido-GTP binding to Gir was not affected by cross-linking indicating that like other G-Proteins, Gir may also have subunits and that cross-linking of Gir with its putative subunits(s) does not block GTP-binding and phosphorylation sites.
Varma
T K
TK
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555.
Singh
U S
US
Srivastava
S K
SK
D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D011257:Pregnancy Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
C021961:8-azidoguanosine triphosphate; D001386:Azides; D003432:Cross-Linking Reagents; D011257:Pregnancy Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D019204:GTP-Binding Proteins