Modification of Adipocyte Membrane Adenylyl Cyclase Activity by NAD: Evidence against NAD-Induced Endogenous ADP-Ribosylation of Gsα Protein

Abstract

Treatment of saponin-permeabilized adipocytes with NAD enhanced adenylyl cyclase activity stimulated by GTγS, [Al/F4]−, isoproterenol, and forskolin in membrane fractions and potentiated isoproterenol-induced cAMP accumulation in whole cells. In parallel, when permeabilized adipocytes were incubated with [32P]NAD, there was significant incorporation of [32P]ADP-ribose in a 44-kDa acceptor membrane protein. This reaction was inhibited by l-arginine and was enhanced by the addition of GTPγS. Surprisingly, this 44-kDa protein could not be identified as Gs protein: (1) It was not recognized by Gsα specific antibody; (2) it did not comigrate with the major cholera toxin substrates in either 10% SDS-PAGE or two-dimensional electrophoresis; (3) a pretreatment of adipocytes with NAD did not decrease cholera toxin-mediated ADP-ribosylation of Gsα proteins on membrane fractions. Our results indicate that NAD did not induce endogenous ADP-ribosylation of Gsα in permeabilized rat adipocytes but nonetheless modified the adenylyl cyclase response.