The chemotactic response of the human melanoma cell line A2058 to type IV collagen is transduced by a pertussis toxin (PT)-sensitive G protein. However, the specific identity of the G protein involved is not known, nor are the events subsequent to G protein activation which result in pseudopodial protrusion and cellular translocation. A method has been devised for the isolation and analysis of pseudopods from A2058 cells which have been stimulated with type IV collagen. Using polycarbonate filters with extremely small pore sizes (1 mum) in a chemotaxis assay, pseudopods formed in response to attractant were isolated from the filters with SDS containing buffer, and analyzed by Western immunoblotting with a panel of antibodies specific for subclasses of Galpha proteins. Results to date indicate that pseudopods formed after type IV collagen stimulation contain a PT sensitive G protein similar (or identical) to human Galphai1 and Galphai2 in the COOH-terminus, but different from both in another, more central region. More specific identification of the G protein in pseudopods will require 2 dimensional gel electrophoresis followed by Western blotting, and/or purification of the G protein from these extracts. In addition, progress has been made in the subcloning and sequencing of cDNAs encoding Galpha subunits isolated from a lambdagt11 library of A2058. Partial sequence information on one insert has confirmed it to be the human Galphai2-encoding cDNA. Many other Galphai1- and Galphai2-like subclones are available for sequence analysis, or possibly for probing with specific antibody in the lambdagt11 expression vector, to search for the relevant one in type IV collagen-mediated chemotaxis.
