Selective formation of G(s alpha)-MHC I complexes after desensitization ofhuman platelets with iloprost

Prolonged treatment of human platelets with the adenylate cyclase-stimulati ng prostacyclin analog iloprost leads to reduction in cAMP formation. Previ ous studies have demonstrated that this may be ascribed to modification of both receptor and G(s alpha) function rather than of the catalytic componen t of adenylate cyclase [Mollner, S., Deppisch, H. & Pfeuffer, T. (1992) Eur . J. Biochem. 210, 539-544]. Iloprost-induced desensitization was accompani ed by the formation of a G(s alpha)-containing 90-kDa product in membranes treated with the bifunctional cross-linker 1,6-bismaleimidohexane. The cAMP -inducing prostanoid PGD(2), which does not promote desensitization, did no t cause formation of the 90-kDa species either. The long-term effect of the common G-protein activator [AlF4](-) on human platelet adenylate cyclase w as shown in many respects to be comparable with that of iloprost. However, [AlF4](-) treatment also failed to induce the 90-kDa species, showing that different mechanisms of desensitization were operating. Treatment of the cr oss-linked 90-kDa complex with PNGase F demonstrated the glycoprotein natur e of the G(s alpha)-associated component. The 90-kDa cross-linked product w as purified by consecutive immunoaffinity chromatography and preparative PA GE to apparent homogeneity. Analysis of the purified protein by MS suggeste d that, besides G(s alpha), the heavy chain of MHC I (HLA-A2) was part of t he complex. This was confirmed by coprecipitation of G(s alpha) by the mono clonal anti-(MHC I) antibody W6/32.