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 (hPAFR), we used the following mutants: a truncated mutant in the C-terminal tail of the receptor (Cys317 --> Stop) and mutations in the (D/N)P(X)2,3Y motif (Asp289 --> Asn,Ala and Tyr293 --> Phe,Ala). Chinese hamster ovary cells expressing the Cys317 --> Stop mutant exhibited a marked reduction in their capacity to internalize PAF, suggesting the existence of determinants important for endocytosis in the last 26 amino acids of the cytoplasmic tail. Substitution of Asp289 to alanine abolished both internalization and G-protein coupling, whereas substitution of Tyr293 to alanine abolished coupling but not internalization. Inhibition or activation of protein kinase C did not significantly affect the internalization process. Receptor sequestration and ligand uptake was, at least in part, blocked by concanavalin A and blockers 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 terminus tail of hPAFR, but not the putative internalization motifs, may be involved in the internalization of hPAFR.
Le Gouill
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C
Immunology Division, Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Parent
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JL
Rola-Pleszczynski
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Stanková
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J
D000818:Animals; D001381:Azepines / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002451:Cell Compartmentation / Q000187:drug effects; D006224:Cricetinae; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D014230:Triazoles / Q000378:metabolism
D001381:Azepines; D007295:Inositol Phosphates; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D014230:Triazoles; C039351:platelet activating factor receptor; C052518:WEB 2086; D011493:Protein Kinase C; D019204:GTP-Binding Proteins