Differential phosphorylation, translocation and signaling of the ovine FP receptor isoforms

Abstract

The autacoid prostaglandin F₂(α)alpha (PGF₂(α)) regulates a variety of physiological responses including luteolysis and parturition. The intracellular effects of PGF₂(α) are elicited by activation of FP G-protein coupled receptors. Alternative splice variants of the ovine FP receptor known as FP(A) and FP(B) exist. Compared to the FP(A), the FP(B) has a truncated intracellular carboxy terminus that lacks 46 amino acids. Both FP isoforms couple to and activate similar signaling cascades. The purpose of this research was to elucidate regulatory and functional differences between the FP receptor isoforms. HEK-293 cell lines stably expressing FLAG-epitope tagged receptor isoforms were generated using PCR cloning to circumvent limitations in obtaining antibodies that could recognize both FP receptor isoforms. The amino terminal FLAG-tagged FP(A) or FP(B)-expressing cells were characterized to show expression levels comparable to that of their non-FLAG-tagged FP(A) or FP(B)-expressing counterparts. Functionally, FLAG-FP(A) or FP(B)- and non-FLAG FP(A) or FP(B)-expressing cells were confirmed to be indistinguishable. PGF₂(α) mediated receptor phosphorylation, internalization, and activation of extracellular signal regulated kinases (ERKs) were investigated and differences between the FP isoforms were found. In vivo phosphorylation assays showed that FP(A) - but not FP(B)-receptors are phosphorylated when treated with PGF₂(α). Immunohistochemical techniques were used to study receptor internalization. The FP(A) receptors undergo internalization that is dependent on PGF₂(α), clathrin and dynamin. The internalization of the FPB receptors however, is independent of PGF₂(α) or clathrin but dependent on dynamin. A putative role for phosphatidyl inositol 3-kinase (PI3-K) in the constitutive sub-cellular distribution of the FP(B) receptors is also reported here. Western blotting techniques showed that both FP receptor isoforms, when stimulated by PGF₂(α) , activate ERKs. Furthermore, the activation of ERKs by both FP isoforms is dependent on protein kinase C and src family tyrosine kinases. However, PI3-K contributes to the activation of ERKs by the FP(B), but not the FP(A) receptors, and may serve as a molecular marker to distinguish ERK signaling by the two isoforms. Finally internalization of either FP isoform may not be required for the PGF₂(α)-mediated activation of ERKs however internalization of both FP isoforms attenuates PGF₂(α) mediated activation of ERKs. In conclusion, the differences in signaling reported here will contribute towards understanding differences in physiological responses elicited by PGF₂(α) and provide markers and targets for therapeutical intervention.