Abstract
GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.
Highlights
A Monoclonal Antibody against theRod Outer Segment Guanyl Nucleotide-binding Protein, Transducin, Blocks the Stimulatory and Inhibitory G Proteins of Adenylate Cyclase*
In early experiments designed to testwhether mAb 4A could block GTP-binding proteinsin brain, we noticed that when synaptic membranes were incubated for a few min with mAb4A, the membranes precipitated, whereas in the presence of nonspecific IgG, no precipitation occurred
Since the antibody has no effect on resolved catalytic subunit, we conclude that itaffects adenylate cyclase activity via its interaction with G proteins
Summary
Immunological Cross-reactivity among GTP-binding Proteins-Monoclonal antibody 4A blocks light activation of ROS phosphodiesterase by interfering with G, binding to and activation by rhodopsin (Hamm et al, 1987). In early experiments designed to testwhether mAb 4A could block GTP-binding proteinsin brain, we noticed that when synaptic membranes were incubated for a few min with mAb4A, the membranes precipitated, whereas in the presence of nonspecific IgG, no precipitation occurred. This suggested that mAb 4A may be binding the homologous G proteins and cross-linking the membranes. Fig. 1shows that mAb 4A reacts strongly with at and cross-reacts with ai subtypes and weakly with as.There is no crossreactivity with control proteins Denaturation of these proteins by treatment of the dot blotwith 2%SDS eliminated ae AnBtibloocdkyade of Adenylate Cyclase G Proteins and Under the conditions of this assay, adenylate cyclase activity is inhibited when Gpp(NH)p
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