Binding of GPVI isoforms to CaM. (A) Binding of MBP-GPVI constructs to CaM in vitro. The ability of CaM to bind to MBP-GPVI cytoplasmic domain fusion proteins in vitro was measured by an immunoprecipitation assay. Four MBP constructs were compared, bearing each of the possible combinations of the 2 cytoplasmic domain amino acid substitutions at position 317 or 322. These are named QH (which represents native GPVIa), and QN, LH, and LN (representing native GPVIb). The components of the immunoprecipitates were separated by SDS-PAGE, and the relative content of MBP fusion protein was determined by Western blot with anti-MBP (top panel). Membranes were stripped and reprobed with anti-CaM antibody (bottom panel). (B) Binding of full-length GPVI isoforms to endogenous Dami cell CaM. We measured by immunoprecipitation the amount of rGPVI in lysates of stable Dami cell transfectants that is coprecipitated with endogenous CaM. Equal volumes of the normalized Dami cell lysates were precleared and incubated overnight with 4 μg of monoclonal anti-CaM. The resulting CaM/anti-CaM immune complexes were isolated by adsorption to fresh protein G agarose beads. Immune complexes were eluted from the beads with SDS electrophoresis buffer, and protein constituents were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot assay. The relative amount of rGPVI recovered was determined by Western blot using LJ6.5 (top panel). Membranes were stripped and reprobed with monoclonal anti-CaM to confirm that the amount of CaM present in each sample was equivalent (bottom panel). The transfected Dami cell lines expressed either rGPVIa (center lane) or rGPVIb (right lane). Mock-transfected cells served as a negative control (left lane).