title: Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms.
abstract: The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
authors.forename: A B
authors.initials: AB
authors.lastname: Fawzi
authors.forename: D S
authors.initials: DS
authors.lastname: Fay
authors.forename: E A
authors.initials: EA
authors.lastname: Murphy
authors.forename: H
authors.initials: H
authors.lastname: Tamir
authors.forename: J J
authors.initials: JJ
authors.lastname: Erdos
authors.forename: J K
authors.initials: JK
authors.lastname: Northup
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002417:Cattle; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D037342:Pertussis Toxin; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D013379:Substrate Specificity; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D012243:Rhodopsin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins