Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.
Little is known about the specific domains of G protein beta and gamma subunits which interact with each other and with the alpha subunit. We used site-specific anti-peptide antibodies directed against beta and gamma subunits to investigate domains on beta and gamma subunits involved in alpha subunit interaction. Antibodies included four against the transducin (Gt) beta subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native beta gamma t subunits. Affinity purification of MS and KT antisera on columns of immobilized native Gt yielded antibodies capable of recognizing native beta gamma t subunits. The functional effects of each antibody preparation on alpha t-beta gamma t interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta gamma t subunits in the presence of excess alpha subunits and by testing the inhibition of beta gamma t-dependent ADP-ribosylation of alpha t-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta gamma t which may be directly involved in alpha t-beta gamma t interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta t, and the carboxyl terminus of gamma t.(ABSTRACT TRUNCATED AT 250 WORDS)
Murakami
T
T
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Simonds
W F
WF
Spiegel
A M
AM
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D002417:Cattle; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D037342:Pertussis Toxin; D011489:Protein Denaturation; D014357:Trypsin / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D000906:Antibodies; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D014357:Trypsin; D019204:GTP-Binding Proteins