The goal of this project is to identify and isolate a cDNA(s) encoding the pertussis toxin sensitive G protein(s) operating in signal transduction pathways responsible for tumor cell motility. The human melanoma cell line A2058 is known to have one or more pertussis toxin sensitive G proteins which transduce chemotactic signals initiated by laminin, type IV collagen, and """"""""autotaxin"""""""". In addition, this cell line has been shown to have at least one 40 kDa G protein labeled by ADP ribosylation catalyzed by pertussis toxin. Previous studies with this cell line (Aznavoorian et al., J Cell Biol 1990;110:1427-37) have shown that transduction of signals initiating motility by extracellular matrix proteins differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Following pretreatment with pertussis toxin, chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to type IV collagen was profoundly sensitive to PT, laminin was intermediate in sensitivity, but fibronectin was completely insensitive. Haptotaxis initiated by all three ligands was unaffected by PT. Modulators of the cAMP pathway had no effect on chemotaxis or haptotaxis. Thus, a PT sensitive, non-cAMP pathway is required to stimulate chemotaxis via laminin or type IV collagen, components of the basement membrane. The specific identity of the G protein involved in A2058 motility is not yet known, although its pertussis toxin sensitivity eliminates G, as a possibility. Potential candidates include one of the Gi-""""""""like"""""""" proteins already cloned, or Go, which is abundant in brain tissues (and has also been found in non-neuronal tissues), but whose physiological function is largely unknown; alternatively, an as yet undiscovered G protein could be involved. The present approach to isolating the cDNA(s) encoding the relevant G protein(s) is to screen the A2058 cDNA library with nucleotide probes after making filter lifts of phage plaques. A series of potential G protein encoding human cDNA clones have been isolated.
