Lack of constitutive activation or inactivation of the platelet-activating factor receptor by glutamate substitution of alanine 230.
The platelet-activating factor (PAF) receptor (PAFR) is a G protein-coupled receptor (GPCR) that mediates a diverse array of biological responses to PAF. Recently, we provided evidence that the third intracellular domain (3i) of the rat PAFR (rPAFR) is a critical determinant in its coupling to phosphoinositide phospholipase C (PI PLC)-activating G proteins. In the present study, we assessed the potential role of a conserved alanine in the carboxyl-terminal region of 3i of the rPAFR in rPAFR signaling activity. Previous studies with the m5 muscarinic acetylcholine and human PAF receptors revealed that substitution of a carboxyl-terminal alanine was found to impair receptor-mediated PI PLC activation. Here we report the effects of the analogous nonconservative substitution of glutamate for alanine 230 of the rPAFR (rPAFR A230E) on receptor-mediated agonist binding and PI PLC activation following transient expression of the receptor cDNA. BHK cells transfected with a cDNA encoding the rPAFR A230E exhibited PAF-stimulated increases in inositol phosphate (IP) accumulation with no increase in basal levels of IPs. PAF-stimulated IP production in rPAFR transfectants was dependent on the amount of DNA transfected, although PAF provoked a larger increase in IPs in rPAFR transfectants than in rPAFRA230E transfectants in cells transfected with equal amounts of receptor cDNA. This latter finding apparently reflects differences in the transfection efficiency or expression of the wild-type and rPAFR A230E cDNAs because PAF produced indistinguishable effects on IP accumulation in rPAFR and rPAFR A230E transfectants expressing equivalent numbers of receptors. These results provide evidence for a nonconserved role of this conserved alanine in coupling of group I GPCRs to PI PLC-activating G proteins and also suggest that this residue has differential roles in regulating expression and signaling by rat and human PAFRs.
Carlson
S A
SA
Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242, USA.
Chatterjee
T K
TK
Fisher
R A
RA
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D007295:Inositol Phosphates / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D010972:Platelet Activating Factor / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
D007295:Inositol Phosphates; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C039351:platelet activating factor receptor; D018698:Glutamic Acid; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D000409:Alanine