RGS4 inhibits platelet-activating factor receptor phosphorylation and cellular responses

To define the role of regulators of G-protein signaling (RGS) in chemoattra ctant-mediated responses, RGS4 and the receptors for platelet-activating fa ctor (PAFR), formylated peptides (FR), or interleukin-8 (CXCR1) were stably coexpressed in a rat basophilic leukemia (RBL-2H3) cell line. The data dem onstrate that RGS4 inhibited responses by PAFR (i.e., phosphoinositide (PI) hydrolysis, Ca2+ mobilization) but not by FR or CXCR1. An N-terminal 33 am ino acid deletion mutant of RGS4 (Delta RGS4), deficient in GAP (GTPase act ivating protein) activity and plasma membrane localization, had no effect o n either PAFR, FR, or CXCR1. RGS4, but not Delta RGS4, also blocked phospho rylation of PAFR by platelet-activating factor (PAF) and, unexpectedly, by phorbol 12-myristate 13-acetate (PMA); it also blocked cross-phosphorylatio n by formylmethionylleucylphenylalanine (fMLP). A point mutant of RGS4 (N88 S), deficient in GAP activity but not membrane localization, partially bloc ked PAFR phosphorylation but had no effect on PAFR-mediated PI hydrolysis a nd Ca2+ mobilization. Truncation of the cytoplasmic tail of PAFR (mPAFR) re sulted in a loss of its susceptibility to inhibition by RGS4. Taken togethe r, the data indicate that of the receptors studied, RGS4 selectively inhibi ted responses to PAFR, which preferentially couples to Gq. At the level of expression studied, RGS4 did not inhibit FR or CXCR1 which activates Gi to transduce cellular signals. Since the tail-deleted mutant of PAFR was not a ffected by RGS4, and RGS4 blocked homologous as well as heterologous phosph orylation of this receptor, it is possible that RGS4 interferes sterically with the cytoplasmic tail of PAFR. Thus, in addition to stimulating the GTP ase activity of G alpha, RGS4 prevents G protein activation by PAFR and the homologous and heterologous phosphorylation of this receptor.