Conserved transducer coupling but different effector linkage upon expression of the myeloid fMet-Leu-Phe receptor in insulin secreting cells.

Abstract

In neutrophils fMet-Leu-Phe activates phospholipase C via a pertussis toxin sensitive G-protein and induces granule secretion. We have transfected a human cDNA sequence encoding the tMet-Leu-Phe receptor into the insulin secreting cell line RINm5F to study receptor-effector coupling with special regard to secretion. Stable overexpression resulted in membrane hyperpolarization, reduction of cAMP accumulation and inhibition of insulin secretion upon exposure of cells to fMet-Leu-Phe with EC50 values in the pmol range. As in the neutrophil, nanomolar concentrations of ligand induced membrane depolarization and activation of phospholipase C, with subsequent mobilization and influx of calcium. In permeabilized cells the inhibitory effect of fMet-Leu-Phe on secretion was partially retained indicating a direct action of the fMet-Leu-Phe receptor on exocytosis. Pertussis toxin abolished the effects of fMet-Leu-Phe. Our results suggest conserved coupling from fMet-Leu-Phe receptor to pertussis toxin sensitive transducers analogous to the mechanism in neutrophils. However, the net biological effect of receptor activation is determined by additional factors intrinsic to the host cell.

The Synthetic Peptide Trp-Lys-Tyr-Met-Val-Met-NH2 Specifically Activates Neutrophils through FPRL1/Lipoxin A4 Receptors and Is an Agonist for the Orphan Monocyte-expressed Chemoattractant Receptor FPRL2

“…Stable Overexpression of FPR, FPRL1, and FPRL2 in RINm5F Cells-The stable expression of FPR in the insulin-secreting cell line RINm5F has been previously described (29). The FPR-expressing RINm5F cells expressed ϳ1.4 ϫ 10 6 receptors/cell.…”

Phosphorylation of Key Serine Residues Is Required for Internalization of the Complement 5a (C5a) Anaphylatoxin Receptor via a β-Arrestin, Dynamin, and Clathrin-dependent Pathway

“…RINm5F cells that stably expressed the mutant receptors C5aR-A 314,317,327,332 and C5aR-A 334,338 were obtained by transfecting cells with 1 g of pPUR plasmid (Clontech) and 20 g of CDM8 mutant receptors as described previously (24). DNA-mediated gene transfer into RINm5F cells was performed by electroporation.…”

Lipopolysaccharide-Induced Granule Mobilization and Priming of the Neutrophil Response toHelicobacter pyloriPeptide Hp(2-20), Which Activates Formyl Peptide Receptor-Like 1

“…To study the presence of FPRL1 in subcellular compartments, a polyclonal anti-FPRL1 antibody was used. The antibody specificity was tested by Western blot analysis with lysates of RINm5F cells overexpressing either FPR or FPRL1 (13,33). A major protein with a molecular mass of about 45 kDa was detected in the lysate of RINm5F cells expressing FPRL1 (Fig.…”