Selective formation of Gsalpha-MHC I complexes after desensitization of human platelets with iloprost.

Publisher: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Country of Publication: England NLM ID: 0107600 Publication Model: Print Cited Medium: Print ISSN: 0014-2956 (Print) Linking ISSN: 00142956 NLM ISO Abbreviation: Eur J Biochem Subsets: MEDLINE

Publication: -2004: Oxford, UK : Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies
Original Publication: Berlin, New York, Springer.

Adenylyl Cyclases/*metabolism ; Blood Platelets/*drug effects ; GTP-Binding Protein alpha Subunits, Gs/*metabolism ; HLA-A2 Antigen/*metabolism ; Iloprost/*pharmacology; Chromatography, Affinity ; Cross-Linking Reagents ; Epoprostenol/analogs & derivatives ; GTP-Binding Protein alpha Subunits, Gs/isolation & purification ; HLA-A2 Antigen/isolation & purification ; Humans ; Platelet Aggregation/physiology ; Platelet Aggregation Inhibitors/pharmacology ; Precipitin Tests

Prolonged treatment of human platelets with the adenylate cyclase-stimulating prostacyclin analog iloprost leads to reduction in cAMP formation. Previous studies have demonstrated that this may be ascribed to modification of both receptor and Gsalpha function rather than of the catalytic component of adenylate cyclase [Mollner, S., Deppisch, H. & Pfeuffer, T. (1992) Eur. J. Biochem. 210, 539-544]. Iloprost-induced desensitization was accompanied by the formation of a Gsalpha-containing 90-kDa product in membranes treated with the bifunctional cross-linker 1,6-bismaleimidohexane. The cAMP-inducing prostanoid PGD2, which does not promote desensitization, did not cause formation of the 90-kDa species either. The long-term effect of the common G-protein activator [AlF4]- on human platelet adenylate cyclase was 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 cross-linked 90-kDa complex with PNGase F demonstrated the glycoprotein nature of the Gsalpha-associated component. The 90-kDa cross-linked product was purified by consecutive immunoaffinity chromatography and preparative PAGE to apparent homogeneity. Analysis of the purified protein by MS suggested that, besides Gsalpha, the heavy chain of MHC I (HLA-A2) was part of the complex. This was confirmed by coprecipitation of Gsalpha by the monoclonal anti-(MHC I) antibody W6/32.

0 (Cross-Linking Reagents)
0 (HLA-A2 Antigen)
0 (Platelet Aggregation Inhibitors)
DCR9Z582X0 (Epoprostenol)
EC 3.6.5.1 (GTP-Binding Protein alpha Subunits, Gs)
EC 4.6.1.1 (Adenylyl Cyclases)
JED5K35YGL (Iloprost)

Date Created: 19990123 Date Completed: 19990305 Latest Revision: 20190620

20231215

10.1046/j.1432-1327.1999.00022.x

9914489