Identification of a Novel Corticotropin-Releasing Hormone Type 1β-Like Receptor Variant Lacking Exon 13 in Human Pregnant Myometrium Regulated by Estradiol-17β and Progesterone

Abstract

Two types of CRH receptors mediate the diverse biological functions of CRH and CRH-related peptides. The type 1 CRH-R (CRH-R1) is extensively targeted by pre-mRNA splicing mechanisms that give rise to multiple mRNA splice variants. RT-PCR amplification of CRH-R1 sequences from human myometrium yielded cDNAs that encode a novel CRH-R1 splice variant with structural characteristics identical with CRH-R1β except a 14-amino acid deletion in the seventh transmembrane domain characteristic of the CRH-R1d. Transient expression of the hybrid CRH-R1 variant (CRH-R1β/d) in human embryonic kidney 293 cells revealed primarily intracellular expression, although some plasma membrane protein expression was also detectable. CRH bound to CRH-R1β/d with affinity comparable with the CRH-R1β; however, it was unable to stimulate adenylyl cyclase or other second messengers. Using a semiquantitative RT-PCR assay, CRH-R1β/d mRNA transcript was detected in human pregnant, but not nonpregnant, myometrium as early as 31 wk of gestation. Furthermore, in human pregnant myometrial cells, the relative expression of CRH-R1β and CRH-R1β/d mRNA appeared to be regulated by steroids; CRH-R1β/d mRNA expression was increased by estradiol-17β, whereas CRH-R1β mRNA levels were increased by progesterone. Progesterone also substantially increased CRH-R1α mRNA levels and cellular responsiveness to CRH as determined by increased agonist binding and cAMP production as well as resistance to CRH-R heterologous desensitization by phorbol esters. These results provide novel evidence for distinct patterns of CRH-R1 splicing and identify specific steroid-mediated regulation of CRH-R1 variant expression, which might be important for modulating CRH actions during human pregnancy and labour.