GTP-binding trimeric proteins have been implicated in signal transduction from receptors in the cell membrane to intracellular effectors and ion channels in a variety of cells (1-5). The mechanism involves signal-induced G-protein activation initiated by an exchange of GDP for GTP on the ␣ subunit of the protein (5-7). Subsequent GTPase activity of the G␣ subunit converts the activated G-proteins into their inactive, GDPbound state (7). Activation of G-proteins has been induced experimentally by stimulation of G-protein-coupled receptors in the cell membrane (2,3,8,9). Evidence indicating activation of G-proteins in response to membrane depolarization were previously observed in brain stem synaptoneurosomes (10 -12).G␣ o -proteins are widely distributed in the central nervous system (15-17). Three subtypes showing marked homology but exhibiting functional differences have been identified (13,14,18). The G␣ o1 subtype appears to be involved in the coupling of muscarinic receptors to Ca ϩ2 channels, and the G␣ o2 subtype mediates inhibition of Ca ϩ2 current activated by somatostatin receptors (19). The function of the G␣ o3 subtype is not clear (14). Phospholipase C activation mediated by activation of G oproteins has been demonstrated in the cell-free system (20), and G o -protein-mediated activation of protein kinase C has been observed in Chinese hamster ovary (CHO) cells (21).In the present study, in situ photoaffinity labeling with [␣ 32 P]GTPAA 1 (22, 23) indicated a depolarization-induced accelerated exchange of GDP for [␣ 32 P]GTPAA in G␣ o1 -and G␣ o3 -proteins, implying a depolarization-induced activation of these G o -proteins. [␣ 32 P]-GTPAA was introduced into transiently permeabilized synaptoneurosomes as described before (10). Unlike the endogenously bound guanine nucleotides, [␣ 32 P]GT-PAA, covalently bound to G␣-proteins by photoaffinity labeling, was not displaced during SDS-polyacrylamide gel electrophoresis, providing a possible tool for identification of in situ activated G-proteins.In view of findings indicating a reciprocal influence of depolarization-induced activation of VGSC and uncoupling of Gproteins from muscarinic receptors (12, 24, 25), we examined the possibility that VGSC can be involved in depolarizationinduced activation of G␣ o -proteins. Our results indicated that depolarization-induced activation of G o -proteins can be prevented by preventing VGSC activation. In addition, in depolarized brain-stem and cortical synaptoneurosomes, the ␣ subunit of VGSC cross-linked most efficiently with G␣ o -proteins. In isolated synaptoneurosomal membranes, VGSC-␣ subunit cross-linked most efficiently with GDP␤S-bound rather than GTP␥S-bound G␣ o -proteins. These findings suggest repeated

GTP-binding protein(s) recognized by antibodies against the ␣-subunits of G i -and G o -proteins were detected in crude nuclei isolated from rat brain stem and cortex. Immunohistochemical staining indicated that in the cortex these proteins are perinuclear, or are embedded in the nuclear membrane. Evidence is presented for an endogenous ADP-ribosylation of these proteins, which competes with their PTX-catalyzed ADP-ribosylation. The endogenous reaction has the characteristics of nonenzymatic ADP-ribosylation of cysteine residues, known to involve NAD-glycohydrolase activity. In vitro experiments showed that the ␣-subunit of G o -proteins in the cell membrane also acts as a substrate of this endogenous ADP-ribosylation. The in situ effect of membrane depolarization on the nuclear GTP-binding proteins may be attributable to their depolarization-induced endogenous ADP-ribosylation, suggesting a novel signaling mechanism in neuronal cells in the central nervous system. NAD is the substrate for enzymes that catalyze ADP-ribosylation, i.e. cleavage of the bond between nicotinamide and ribose and the transfer of ADP-ribose to nucleophilic acceptors (1). ADP-ribosylation represents a mechanism for post-translational modification of proteins. Among the known acceptors for ADP-ribose are GTP-binding proteins in cell membranes (2). ADP-ribosyltransferases that catalyze ADP-ribosylation of arginine residues in GTP-binding proteins (similar to cholera toxin) (2) have been detected in eukaryotic cells (3). An endogenous ADP-ribosylation of cysteine residues of membrane Gproteins (similar to pertussis toxin (PTX) 1 -catalyzed ADP-ribosylation) (2) has been suggested to occur in erythrocytes (4,5).In this report we present evidence for an endogenous ADPribosylation of cysteine residues in GTP-binding protein(s) in the nuclei of cells in rat brain stem and cortex. These proteins also act as substrates of PTX-catalyzed ADP-ribosylation and react with antibodies against the ␣-subunits of G i -and G oproteins (G␣ i -and G␣ o -proteins). Unlike these membrane Gproteins, however, the nuclear GTP-binding proteins are apparently modified by a nonenzymatic ADP-ribosylation of cysteine residues, which competes with their PTX-catalyzed ADPribosylation. Also, unlike membrane G-proteins, the nuclear GTP-binding proteins are not extractable by detergents, nor are they activated by membrane depolarization (6 -9). The evidence presented here is consistent with a depolarization-induced ADP-ribosylation of these nuclear GTP-binding proteins, suggesting a novel signaling mechanism in neuronal cells in the central nervous system. MATERIALS AND METHODSReagents-Nicotinamide-adenine dinucleotide (grade I), adenosine triphosphate (grade I), GDP␤S, GTP␥S, tetrodotoxin, dithiothreitol (DTT), azidoaniline, sodium nitroprusside, 3-aminobenzamide (3-AB), and thymine were all purchased from Sigma. PTX and the A-protomer of PTX (ADP-ribosyltransferase) were purchased from List Biological Laboratories. [ 14 C]NAD (57 mCi/mmol) was purchased from Amersham Corp...

Synaptoneurosomes (1-3 microm in diameter), prepared from rat brain stem or brain cortex, were fused with liposomes, producing a high yield of giant synaptosomes (10-60 microm in diameter). Single channel currents were measured by using the cell-attach patch-clamp technique. The membrane of the majority of these giant synaptosomes retained the cell membrane selective permeability. However, nonpermeating molecules, such as guanine nucleotides and antibodies directed against GTP-binding region in the alpha-subunit of trimeric GTP-binding proteins, were trapped in the giant synaptosomes during their preparation. Activation of Go proteins was assayed in high [K(+)]-depolarized giant synaptosomes, indicating the advantage of this preparation for tracing signal-transduction mechanisms in stimulated synaptic membranes. Stimulation-induced interactions between membrane proteins, either native or reconstituted, can be studied in the giant synaptosomes.