Activation and regulation of platelet-activating factor receptor: role of G(i) and G(q) in receptor-mediated chemotactic, cytotoxic, and cross-regulatory signals.
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of G(i) and G(q) in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing G alpha(i3) (PAFR-G alpha(i3)) or G alpha(q) (PAFR-G alpha(q)) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-G alpha(i3), or PAFR-G alpha(q) was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of beta-hexosaminidase. PAFR-G alpha(i3) and PAFR-G alpha(q) mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-G alpha(i3), but not PAFR-G alpha(q), mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated beta arrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca2+ mobilization to PAF by all the receptors but only PAFR-G alpha(i3) activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G(i) exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G(i) and G(q) activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G(i), the data suggest that differential activation of both G(i) and G(q) by PAFR likely mediate specific as well as redundant signaling pathways.
Brown
Stephan L
SL
Department of Biochemistry, Meharry Medical College, Nashville, TN 37208, USA.
Jala
Venkatakrishna R
VR
Raghuwanshi
Sandeep K
SK
Nasser
Mohd W
MW
Haribabu
Bodduluri
B
Richardson
Ricardo M
RM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D002633:Chemotaxis; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010972:Platelet Activating Factor / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D023062:Receptors, Interleukin-8A / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D023062:Receptors, Interleukin-8A; C039351:platelet activating factor receptor; D037342:Pertussis Toxin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium