Calmodulin binding distinguishes between beta gamma subunits of activated G proteins and transducin.
The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.
Mangels
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LA
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Neubig
R R
RR
Hamm
H E
HE
Gnegy
M E
ME
D000067956:Adenylyl Cyclase Inhibitors; D000535:Aluminum / Q000494:pharmacology; D000077410:Aluminum Chloride; D017607:Aluminum Compounds; D000818:Animals; D001923:Brain Chemistry; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D015293:Transducin / Q000378:metabolism*
D000067956:Adenylyl Cyclase Inhibitors; D017607:Aluminum Compounds; D002147:Calmodulin; D002712:Chlorides; D046911:Macromolecular Substances; D015636:Magnesium Chloride; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000077410:Aluminum Chloride; D012969:Sodium Fluoride; D000535:Aluminum; D019204:GTP-Binding Proteins; D015293:Transducin