Characterization of human phagocytic cell receptors for C5A and platelet activating factor expressed in Xenopus oocytes.
Platelet activating factor (PAF) and the active cleavage product of the fifth component of complement, C5a, are potent anaphylotoxins and mediators of inflammation. Both substances engage distinct guanine nucleotide binding regulatory protein-coupled receptors on a variety of cell types, thereby activating a signaling cascade that results in the mobilization of intracellular calcium stores, and in functional responses such as neutrophil chemotaxis and smooth muscle contraction. Little is known about the structure of PAF and C5a receptors or about the intracellular signaling pathways used by them. We have used the Xenopus oocyte expression system to demonstrate acquired C5a and PAF receptor activity in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 differentiated with dibutyryl cAMP. Activity was determined by measuring acquired ligand-dependent efflux of intracellular 45Ca2+ and by measuring ligand-activated transmembrane currents in voltage clamped oocytes. C5a receptor activity was confined to a single sharp peak in sucrose gradient fractionated RNA that corresponded to a transcript size of 2 kb. In contrast, PAF receptor activity was broadly distributed in size fractionated RNA from 3.5 to 6 kb. This suggests that multiple transcripts of different sizes may encode a functional PAF receptor. Both ligands activated their respective receptor in a concentration-dependent and a structure-dependent manner. The acquired C5a-dependent calcium efflux activity was inhibited by pertussis toxin whereas the PAF receptor activity was not, suggesting that the two receptors couple to different G-proteins. These data establish the Xenopus oocyte as a model system for studying the molecular and functional properties of the C5a receptor and the PAF receptor(s) of phagocytic cells.
Murphy
P M
PM
Bacterial Diseases Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Gallin
E K
EK
Tiffany
H L
HL
D000818:Animals; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008845:Microinjections; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009865:Oocytes; D037342:Pertussis Toxin; D010586:Phagocytes / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics; D044087:Receptor, Anaphylatoxin C5a; D011956:Receptors, Cell Surface / Q000235:genetics*; D011951:Receptors, Complement / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014982:Xenopus laevis
D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D044087:Receptor, Anaphylatoxin C5a; D011956:Receptors, Cell Surface; D011951:Receptors, Complement; D043562:Receptors, G-Protein-Coupled; D010566:Virulence Factors, Bordetella; C039351:platelet activating factor receptor; D009240:N-Formylmethionine Leucyl-Phenylalanine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium