Delayed Reversal of Shape Change in Cells Expressing FPB Prostanoid Receptors: POSSIBLE ROLE OF RECEPTOR RESENSITIZATION

Hiromichi Fujino,Kristen L Pierce,Dinesh Srinivasan,Charles E Protzman,Achim H Krauss,David F Woodward,John W Regan

doi:10.1074/jbc.m003467200

Abstract

Prostaglandin F(2 alpha) (PGF(2 alpha)) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FP(A) and FP(B). As compared with the FP(A) isoform, the FP(B) isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FP(A). We recently found (Pierce, K. L., Fujino, H., Srinivasan, D., and Regan, J. W. (1999) J. Biol. Chem. 274, 35944-35949) that stimulation of both isoforms with PGF(2 alpha) leads to activation of a Rho signaling pathway, resulting in tyrosine phosphorylation of p125 focal adhesion kinase, formation of actin stress fibers, and cell rounding. Although the activation of Rho and subsequent cell rounding occur at a similar rate for both isoforms, we now report that following the removal of PGF(2 alpha) the reversal of cell rounding is much slower for cells expressing the FP(B) isoform as compared with the FP(A) isoform. Thus, in HEK-293 cells that stably express the FP(A) isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FP(B)-expressing cells there is essentially no reversal even after 2 h. Similarly, the disappearance of stress fibers and dephosphorylation of p125 focal adhesion kinase following removal of agonist are much slower in FP(B)-expressing cells than in FP(A)-expressing cells. The mechanism of this differential reversal appears to involve a difference in receptor resensitization following the removal of agonist. Based upon whole cell radioligand binding, agonist-induced stimulation of inositol phosphate formation, and mobilization of intracellular Ca(2+), the FP(B) isoform resensitizes more slowly than the FP(A) isoform. These findings suggest that the carboxyl terminus of the FP(A) is critical for resensitization and that the slower resensitization of the FP(B) isoform leads to prolonged signaling. This differential signaling distinguishes the FP(A) and FP(B) receptor isoforms and could be important toward understanding the physiological actions of PGF(2 alpha).

Highlights

Prostaglandin F2␣ (PGF2␣) receptors are G-proteincoupled receptors consisting of two alternative mRNA splice variants, named FPA and FPB
Besides coupling to Gq and the phosphoinositides signaling pathway, we have found that the FPA and FPB prostanoid receptor isoforms activate Rho, a member of the Ras family of small G-proteins, to cause cell rounding, actin stress fiber formation, and tyrosine phosphorylation of p125 focal adhesion kinase (FAK) [7]
Reversal of PGF2␣-induced Cell Rounding Is Slower for Cells Expressing the FPB Prostanoid Receptor Isoform than for Cells Expressing the FPA Isoform—Recently, we have shown that treatment of the ovine FPA and FPB prostanoid receptor isoforms with PGF2␣ leads to changes in cellular morphology consisting of the retraction of filopodia, cell rounding, and a cobblestone appearance of cell aggregates [1]

Summary

The abbreviations used are

PGF2␣, prostaglandin F2␣; FAK, focal adhesion kinase; PKC, Ca2ϩ-dependent protein kinase; LPA, lysophosphatidic acid; TRITC, Texas Red isothiocyanate; MES, 2-(N-morpholino)ethanesulfonic acid Based on the results of radioligand binding, agonist-stimulated inositol phosphate accumulation, and mobilization of intracellular calcium, it appears that this slower reversal is related to slower resensitization of the FPB isoform following agonist-induced desensitization. While both isoforms initially desensitize to a similar extent following treatment with PGF2␣, the FPB resensitizes more slowly than the FPA. We hypothesize that the slower resensitization of the FPB isoform is associated with prolonged signaling following the removal of agonist

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION