Cloning and characterization of novel isoforms of the human receptor for prostaglandin F2 alpha

Abstract

Prostaglandin F2 alpha (PGF₂α) regulates physiological responses including lowering of intraocular pressure, luteolysis, and parturition. FP prostanoid receptors are GPCRs mediating the actions of PGF₂α. mRNA splice variants exist for the ovine FP receptor gene and are designated FP(A) and FP(B). Thus far, receptor heterogeneity for the human FP receptor gene has yet to be established. This dissertation identifies human FP receptor isoforms and their pharmacological significance. Utilizing PCR, a putative human FP(B) (hFP(B)) ortholog from placenta and CX-1 (colon adenocarcinoma) cDNA was identified. These clones produced similar cDNA sequence both containing inverted repeat sequences. However, the mechanism by which the hFP(B) is produced appears different from alternative mRNA splicing and remains unexplained. Pharmacological characterization of the hFP(B) ortholog is ongoing and its significance remains unknown. Additionally, we report the cloning of a FP receptor mRNA splice variant from human heart and placenta cDNA, named human FP sevenless (hFP(S)). The cDNA encoding hFP(S) has a 71 base pair insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and a carboxyl tail. This sequence is identified as a distinct exon localized on the human FP receptor gene. hFP(S) mRNA are expressed in human skeletal muscle, heart and placenta. Immunohistochemical staining showed positive immunoreactivity in vascular endothelium, trophoblast, and decidual cells from placenta. hFP(S) represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is unknown. Pharmacological characterization of hFP(S) demonstrated no significant PGF₂α binding or PGF₂α-mediated inositol phosphate hydrolysis. FLIPR high throughput screening membrane potential assays yielded four potential agonists for hFPS activation that were not observed in (-)293-EBNA cells; oxytocin, PGB₂, AGNA9B9, and AGNA10B10. These potential agonists require further investigation for hFP(S) selectivity and until such data is determined, hFP(S) should continue to be considered an orphan receptor. In conclusion, these studies demonstrate one FP receptor splice variant of the six transmembrane (6TM) nature exists in humans. In addition, preliminary evidence suggests the existence of a hFP(B) receptor ortholog potentially generated from a mechanism other than traditional mRNA splicing and could contribute to the development of colon carcinogenesis.