Isolation and characterization of the corticotropin-releasing factor-related diuretic hormone receptor in Rhodnius prolixus.

Rhodnius prolixus, the vector of human Chagas disease, is a hemipteran insect that undergoes rapid post-feeding diuresis following ingestion of a blood meal that can be up to 10 times its initial body weight. Corticotropin-releasing factor-related diuretic hormone (Rhopr-CRF/DH) and serotonin are neurohormones that are synergistic in increasing rates of fluid secretion by Malpighian tubules during this rapid post-feeding diuresis. A Rhopr-CRF/DH receptor transcript has now been isolated and characterized from fifth instar R. prolixus. The receptor is a family B1 (secretin) G protein-coupled receptor (GPCR) and was deorphaned in a heterologous cellular system using Chinese hamster ovary (CHO) cells stably expressing a promiscuous G-protein (Gα16). This assay was also used to demonstrate the presence of Rhopr-CRF/DH in the haemolymph of R. prolixus in response to blood-gorging. Two additional cell lines were used in this heterologous assay to verify that the cyclic adenosine monophosphate (cAMP) pathway and not the inositol triphosphate (IP3) pathway was stimulated upon activation of the receptor. Lastly, quantitative PCR demonstrated strong receptor expression in digestive tissues, upper Malpighian tubules and reproductive tissues. Identification of the Rhopr-CRF/DH receptor now provides tools for a more detailed understanding into the precise coordination of diuresis and other physiological processes in R. prolixus.

Identification of a novel corticotropin-releasing hormone type 1beta-like receptor variant lacking Exon 13 in human pregnant myometrium regulated by estradiol-17beta and progesterone.

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-R1ß 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-R1ß/d) in human embryonic kidney 293 cells revealed primarily intracellular expression, although some plasma membrane protein expression was also detectable. CRH bound to CRH-R1ß/d with affinity comparable with the CRH-R1ß; however, it was unable to stimulate adenylyl cyclase or other second messengers. Using a semiquantitative RT-PCR assay, CRH-R1ß/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-R1ß and CRH-R1ß/d mRNA appeared to be regulated by steroids; CRH-R1ß/d mRNA expression was increased by estradiol-17ß, whereas CRH-R1ß 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.

On-column refolding of corticotropin-releasing factor receptor 1 extracellular domain by size exclusion chromatography.

Expressing the extracellular domain of corticotropin releasing factor receptor 1 in Escherichia coli usually results in the formation of inclusion bodies. Here we describe the optimization of refolding by applying size exclusion chromatography with a denaturing guanidine hydrochloride gradient and a refolding buffer containing glycerol. Several chromatographic parameters like gradient length, flow rate, sample concentration and chromatography resin characteristics were evaluated. Recovery yields of refolded protein above 50% using a Superdex 200 column demonstrate the usefulness of this method.

Solubilization and biochemical characterization of the human myometrial corticotrophin-releasing hormone receptor.

We have solubilized an active form of the myometrial corticotrophin-releasing hormone (CRH) receptor using 1% w/v digitonin. The solubilized receptor retains its capacity for high-affinity binding as demonstrated by Scatchard analysis, although there was a shift in dissociation constant (Kd) from 83.6 +/- 15-195 +/- 35 pM for the membrane-bound and soluble receptor respectively. There was no difference in the maximum binding site concentrations (Bmax) of 13 +/- 5 and 21.5 +/- 6 fmol/mg protein for the membrane-bound and soluble receptor respectively. Sauvagine unlike CRH had no effect on radiolabeled CRH binding which suggests that the CRH-R2 receptor is not present in the myometrium. The solubilized receptor did not retain guanine-nucleotide sensitivity. The isoelectric focusing (IEF) profile of the human myometrial CRH receptors was significantly different from that of the rat cerebral cortex. Furthermore, solubilization of human myometrial membrane proteins followed by gel filtration and SDS-PAGE revealed a specifically labeled protein with an apparent molecular weight of 42000-47000 kDa. Our results suggest that during solubilization the human myometrial CRH receptor is dissociated from the guanine nucleotide-binding protein (Gs) and that high affinity binding for soluble CRH receptors is not dependent on the coupling of a guanine nucleotide-binding protein.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.