Alanine Exchanges of Polar Amino Acids in the Transmembrane Domains of a Platelet-activating Factor Receptor Generate Both Constitutively Active and Inactive Mutants

Abstract

To determine ligand-binding sites of a platelet-activating factor (PAF) receptor, alanine-scanning mutagenesis was carried out. All 23 polar amino acids in the putative 7-transmembrane (TM) domains of a guinea pig PAF receptor were individually replaced with alanine. The ligand-binding properties of mutant receptors were determined after transient expression in COS-7 cells. Mutants in TM II (N58A, D63A), TM III (N100A, T101A, S104A) and TM VII (D289A) displayed higher PAF-binding affinities than seen with the wild-type receptor. In contrast, mutants in TM V (H188A), TM VI (H248A, H249A, Q252A), and TM VII (Q276A, T278A) showed lower affinities. Representative mutants were then stably expressed in Chinese hamster ovary cells to observe PAF-induced cellular signals (arachidonate release, phosphatidylinositol hydrolysis, adenylyl cyclase inhibition). An N100A mutant with the highest affinity was constitutively active and was responsive to lyso-PAF, an inactive derivative of PAF. One nanomolar PAF induced no signals in low affinity mutants, an EC 50 value for the wild-type receptor. Three histidines (His-188, His-248, His-249) might form a binding pocket for the phosphate group of PAF, since zinc effectively inhibited ligand binding. Based on these results, a three-dimensional molecular model of PAF and its receptor was generated using bacteriorhodopsin as a reference protein.Platelet-activating factor (PAF), 1 is a potent phospholipid mediator with diverse physiological actions on a wide variety of cells and tissues. PAF is thought to play important roles in allergic disorders, inflammation, shock, and some diseases and also to have effects on the reproductive, cardiovascular, and central nervous systems (1-4). Despite the highly hydrophobic structure with a glycerophospholipid skeleton, PAF binds to a cell surface receptor, which was first cloned from a guinea pig lung cDNA library (5). The PAF receptor, with a seven-transmembrane (TM) topology like that of rhodopsin, belongs to a G protein-coupled receptor (GPCR) superfamily. PAF receptor homologs in four mammalian species (guinea pig, human, rat, and mouse) have been reported (6 -12). The PAF receptors couple with various second messenger systems including activation of phospholipase A 2 , C, and D; activation of mitogenactivated protein kinase, phosphatidylinositol 3-kinase, and tyrosine kinases; and inhibition of adenylyl cyclase, thus exerting pleiotropic effects (13-15).To date, a large body of information regarding ligand (agonist and antagonist)-binding characteristics of GPCRs represented by ␤-adrenergic receptors has been obtained (16), and this greatly contributes to effective clinical applications. Various PAF receptor antagonists have been developed as antiallergic and antiinflammatory drugs, but little is known of ligand-binding sites in the cloned PAF receptors. Most GPCR agonists are considered to bind to a hydrophobic core surrounded by seven-TM ␣-helices with electrostatic and hydrophobic force. We designed experiments using alanine-scanning muta...

The Structural Evolution of a P2Y-like G-protein-coupled Receptor

“…This indicates that Asn-137 participates in maintaining the ground state of receptor activity. Our results are in concert with previous studies showing that mutation of Asn (relative position 3.35) in G␣ q /G␣ i -coupled GPCRs such as the platelet-activating factor (PAF) receptor, the bradykinin B2 receptor, and the AT1 angiotensin II receptor induces constitutive activity (26,27).…”

Platelet-activating Factor (PAF) Induces Growth Stimulation, Inhibition, and Suppression of Oncogenic Transformation in NRK Cells Overexpressing the PAF Receptor

“…Using the cloned PAFR and its mutants expressed in Chinese hamster ovary cells, we showed that activation of PAFR leads to inhibition of adenylate cyclase, elevation of intracellular calcium concentration, arachidonic acid liberation, and mitogen-activated protein kinase activation. These intracellular effectors differed in their sensitivities to pertussis toxin (PTX) treatment (16,21).…”

Paf receptor expression in the marsupial embryo and endometrium during embryonic diapause

“…In the tammar, the transmembrane domains had the highest conservation with eutherians and these were also highly conserved among all marsupial species. Based on mutation studies, transmembrane domains 5, 6 and 7 are predicted to be involved in Paf binding and, in particular, three histidine residues are thought to constitute the three-dimensional Paf-binding pocket and were completely conserved in the tammar (Ishii et al 1997). Other regions important for PTAFR function that were conserved in the tammar include the third intracellular loop, which is required for intracellular signalling, and the C-terminal domain, which is phosphorylated before desensitisation of the receptor (Prescott et al 2000).…”

“…The tammar PTAFR gene was highly conserved with that of PTAFR in eutherian species. Furthermore, the seven transmembrane domains were all highly conserved between tammar and all species examined, and this included conservation of the putative Paf-binding pocket (Ishii et al 1997, Prescott et al 2000. These domains are characteristic of a member of the G proteincoupled receptor superfamily and, together with conservation of the regions involved in translation of PTAFR signalling and desensitisation (Ishii & Shimizu 2000, Prescott et al 2000, suggest that the functional role of PTAFR has been conserved in the tammar.…”