A novel 100 kDa protein, localized to receptor-enriched endosomes, is immunologically related to the signal-transducing guanine-nucleotide-binding proteins Gt and Gi.
Antisera raised against the C-terminus decapeptide of the alpha-subunit of the retinal guanine-nucleotide-binding protein (G-protein) transducing (Gt) cross-reacted with the alpha-subunit of the inhibitory G-protein Gi. The same antisera also reacted with a 100 kDa protein (p100) found in rat liver homogenates. The immunoreactivity of both Gt and p100 was specifically inhibited by the immunizing peptide with similar dose-dependencies [concn. causing 50% inhibition (IC50) = 300 ng/ml]. This similarity in inhibition profiles implies that p100 contains within its structure the C-terminal sequence shared by both alpha t and alpha i. Tissue distribution studies demonstrated that p100 was particularly enriched in the liver and kidney, but was also present in other rat tissues, as well as in a number of cell lines tested. In the liver, p100 was found in both the soluble and membrane fractions. The membrane-associated form of p100 was specifically localized to an endosomal fraction (termed D-R), previously shown to be a ligand-free but receptor-enriched subfraction of liver endosomal vesicles. Two-dimensional gel electrophoresis revealed that both the cytosolic and membrane-bound forms of p100 occurred as a series of 100 kDa polypeptides with considerable charge heterogeneity (pI 6-7). Because the C-terminus domains of both alpha t and alpha i facilitate their association with their respective receptors, this region has been functionally assigned as the receptor binding site. Therefore the presence of an immunologically similar region within p100, together with its localization to the receptor-rich endocytic vesicles, suggests that p100 may be a receptor binding protein involved in receptor trafficking.
Traub
L M
LM
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Evans
W H
WH
Sagi-Eisenberg
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000276:immunology; D006056:Golgi Apparatus / Q000737:chemistry; D015151:Immunoblotting; D008099:Liver / Q000737:chemistry / Q000648:ultrastructure; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D008970:Molecular Weight; D015388:Organelles / Q000737:chemistry*; D051381:Rats; D011919:Rats, Inbred Strains; D012374:Rod Cell Outer Segment / Q000737:chemistry; D015398:Signal Transduction
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D019204:GTP-Binding Proteins