Carlson S A, Chatterjee T K, Fisher R A
Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242, USA.
Recept Signal Transduct. 1996;6(2):111-20.
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.
血小板活化因子（PAF）受体（PAFR）是一种G蛋白偶联受体（GPCR），介导对PAF的多种生物学反应。最近，我们提供的证据表明，大鼠PAF受体（rPAFR）的第三个细胞内结构域（3i）是其与磷酸肌醇磷脂酶C（PI PLC）激活型G蛋白偶联的关键决定因素。在本研究中，我们评估了rPAFR的3i羧基末端区域中一个保守丙氨酸在rPAFR信号传导活性中的潜在作用。先前对m5毒蕈碱型乙酰胆碱受体和人PAF受体的研究表明，羧基末端丙氨酸的替代会损害受体介导的PI PLC激活。在此我们报告，在受体cDNA瞬时表达后，将rPAFR的丙氨酸230类似地非保守替换为谷氨酸（rPAFR A230E）对受体介导的激动剂结合和PI PLC激活的影响。用编码rPAFR A230E的cDNA转染的BHK细胞表现出PAF刺激的肌醇磷酸（IP）积累增加，而IP的基础水平没有增加。rPAFR转染细胞中PAF刺激的IP产生取决于转染的DNA量，尽管在用等量受体cDNA转染的细胞中，PAF在rPAFR转染细胞中引起的IP增加比在rPAFR A230E转染细胞中更大。后一发现显然反映了野生型和rPAFR A230E cDNA的转染效率或表达差异，因为PAF对表达等量受体的rPAFR和rPAFR A230E转染细胞中的IP积累产生了难以区分的影响。这些结果为该保守丙氨酸在I型GPCR与PI PLC激活型G蛋白偶联中的非保守作用提供了证据，也表明该残基在调节大鼠和人PAFR的表达和信号传导中具有不同作用。
