Abstract
Islet-activating protein (IAP), one of the pertussis toxins, exerted dual actions on crude membrane preparations from rat C6 glioma cells; an Mr = 41,000 membrane protein was ADP-ribosylated while GTP (and GTP-dependent isoproterenol) activation of membrane adenylate cyclase was enhanced when membranes were incubated with IaP. Both actions of IaP were dependent on the incubation time and the concentrations of NAD and IAP, and were inhibited by nicotinamide; the one action was strictly paralleled by the other in magnitude. Tryptic digestion of the Mr = 41,000 protein was markedly influenced by the presence of guanyl-5'-yl beta-gamma-imidodiphosphate or NaF, the specific ligands of the regulatory component of the adenylate cyclase system. No ADP ribosylation occurred in the membranes prepared from intact C6 cells that had been incubated with IAP, suggesting that the IAP substrate had already been ADP-ribosylated by the intracellular NAD during incubation of the intact cells. Cholera toxin catalyzed ADP ribosylation of other proteins with Mr = 45,000 and 48,000/49,000 (doublet). It is concluded that IAP, added to intact cells or isolated membranes, causes unique modification of the receptor-adenylate cyclase coupling mechanism as a result of ADP ribosylation of the Mr = 41,000 protein which is presumably one of the subunits, other than the cholera toxin substrates, of the guanine nucleotide regulatory component of the cyclase system.
Highlights
Were incubated with IAP.Both actions of IAP were dependent on the incubation time and the concentrations of NAD and IAP, and were inhibited by nicotinamide; the one action was strictly paralleled by the other in magnitude.Tryptic digestion of the Mr = 41,000 protein was markedly influenced by the presence of guanyl-8-yl fi-y-imidodiphosphate or NaF, the specific our recent paper [12] has afforded a clue as to how IAP interactswith cell membranes
It is concluded hanced G T P activation of adenylate cyclase under a variety that IAP, added to intact cells or isolated membranes, of conditions, ( b ) theproteinADP-ribosylated by IAP is causes unique modification of the receptor-adenylate distinct from thceholera toxinsubstrate, ( c )the susceptibility cyclase coupling mechanism as a result of ADP ribo- of the IAP-specific substrate protein to tryptic digestion is sylation of the M, = 41,000 protein which is presumably profoundly affected by the presence of guanine nucleotides, one of the subunits, other than the cholera toxin sub- and (d) theIAPsubstrateprotein is not radiolabeledin strates, of the guanine nucleotide regulatory compo- membranes prepared from IAP-treatedcells, suggesting that nent of the cyclase system
That ADP ribosylation of membrane proteinscatalyzed by IAP in intact cells is responsible for enhancedG T P activation of adenylate cyclase characteristic of membranes prepared from the IAPtreated cells
Summary
C6 cell membranes were first treated with IAP (25 pg/ml) or preactivated cholera toxin (50 pg/ml) in the presence of 0.2 m~ GTP and 1 mM (instead of 10 PM) NAD, and assayed for adenylate cyclase, as described under “Experimental Procedures.” The concentrations of additions during assay were 10 p~ for GTP, Gpp(NH)p, and isoproterenol (Isp),and 10 mM for NaF.
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