STRUCTURAL AND FUNCTIONAL REQUIREMENTS FOR AGONIST-INDUCED INTERNALIZATION OF THE HUMAN PLATELET-ACTIVATING-FACTOR RECEPTOR

The receptor for platelet-activating factor (PAF) is a member of the G -protein-coupled receptor family. To study the structural elements and mechanisms involved in the internalization of human PAF receptor (hPA FR), we used the following mutants: a truncated mutant in the C-termin al tail of the receptor (Cys(317) --> Stop) and mutations in the (D/N) P(X)(2,3)Y motif (Asp(289) --> Asn,Ala and Tyr(293) --> Phe,Ala). Chin ese hamster ovary cells expressing the Cys(317) --> Stop mutant exhibi ted a marked reduction in their capacity to internalize PAF, suggestin g the existence of determinants important for endocytosis in the last 26 amino acids of the cytoplasmic tail. Substitution of Asp(289) to al anine abolished both internalization and G-protein coupling, whereas s ubstitution of Tyr(293) to alanine abolished coupling but not internal ization. Inhibition or activation of protein kinase C did not signific antly affect the internalization process, Receptor sequestration and l igand uptake was, at least in part, blocked by concanavalin A and bloc kers of endocytosis mediated by clathrin-coated pits, Our data suggest that the internalization of a G-protein-coupled receptor and coupling to a G-protein can be two independent events. Moreover, the C terminu s tail of hPAFR, but not the putative internalization motifs, may be i nvolved in the internalization of hPAFR.