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.

Biomimetic screening of class-B G protein-coupled receptors.

The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF(1)R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF(1) receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure-activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC(50) = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.

An oxidation resistant radioligand for corticotropin-releasing factor receptors.

The methionine residues in Tyr-corticotropin-releasing factor (CRF) and Tyr-sauvagine radioligands are subject to oxidation, which renders them biologically inactive. Therefore [Tyr(0,) Gln(1,) Leu(17)]sauvagine (YQLS), in which the methionine was replaced with leucine was synthesized and labeled with (125)Iodine using chloramine-T. Mass spectroscopy revealed that chloramine-T-treatment did not oxidize YQLS. (125)I-YQLS bound with high affinity to cells expressing the murine CRF receptor 1 (CRFR1), CRF receptor 2 (CRFR2), and the mouse brain regions known to express both CRF receptors. (125)I-YQLS chemically cross-linked to CRFR1. In conclusion, (125)I-YQLS is oxidation-resistant, high affinity radioligand that can be chemically cross-linked to the CRF receptors.

Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF) photoaffinity labeling studies on CRF receptor, type 1 (CRFR1).

Novel photoactivatable antagonists of human/rat corticotropin-releasing factor (h/rCRF) have been synthesized and characterized. The N-terminal amino acid D-phenylalanine in astressin ¿cyclo(30-33) [D-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12-41)¿, a potent CRF peptide antagonist, was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. Additionally, His32 of astressin was substituted by either alanine or tyrosine for specific radioactive labeling with 125I at either His13 or Tyr32, respectively. The photoactivatable CRF antagonists were tested for their ability to displace 125I-labeled Tyr0 ovine CRF ([125I-labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y-79 retinoblastoma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB-cyclo(30-33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF-(13-41) (compound 1) was found to bind with higher affinity to rat or human CRFR1 when compared with ATB-cyclo(30-33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF-(13-41) (compound 2) and exhibited higher inhibition of oCRF-stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells) or Y-79 cells. A highly glycosylated, 66-kDa protein was identified with SDS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable 125I-labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analogs could be displaced from the receptor by their corresponding nonlabeled form, but not other unrelated peptides such as vasoactive intestinal peptide. The observed molecular size of the receptor was in agreement with the size of CRFR1 found in rat pituitary (66 kDa), but was significantly larger than the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).

Cloning and characterization of a short variant of the corticotropin-releasing factor receptor subtype from rat amygdala.

We have identified and characterized a cDNA encoding a novel isoform of the corticotropin-releasing factor (CRF) receptor, referred to as CRF2alpha-tr, from the rat amygdala cDNA library. The nucleotide sequence of the cloned cDNA has a structure of an alternatively spliced form of the CRF2alpha receptor, which contains unspliced introns 6 and 7 in the message, and encodes a 236-amino-acid truncated protein that comprises three unique transmembrane domains. Northern blot analysis shows that the CRF2alpha-tr receptor is more strongly expressed in the rat amygdala, thalamus, and hypothalamus than the intact CRF2alpha receptor. Western blot analysis also reveals that the CRF2alpha-tr protein can be expressed in transfected COS-7 cells as well as CRF2alpha. Furthermore, this receptor binds rat/human CRF with almost the same low affinity (Kd = 12.7 nM) as the CRF2alpha and without accumulation of intracellular cAMP. Interestingly, it does not bind sauvagine or rat urocortin. These findings suggest that this truncated CRF receptor is the major isoform of CRF2alpha receptor mRNA transcripts in the amygdala and would mediate some functions of CRF pathways in the central nervous system.

Identification of amino acids in the N-terminal domain of corticotropin-releasing factor receptor 1 that are important determinants of high-affinity ligand binding.

The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists: one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg76 or Asn81 but not Gly83 of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.

CRF2 alpha and CRF2 beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues.

We have recently described the cloning and characterization of a novel corticotropin-releasing factor receptor subtype (CRF2) from rat brain that exists in two alternatively spliced forms, CRF2 alpha and CRF2 beta. These forms differ in their N-terminal coding sequence which results in the production of two distinct receptors of 411 and 431 amino acids, respectively. To assess whether these two forms might represent distinct targets for CRF action, RNase protection and in situ hybridization studies were performed using specific N-terminal cRNA probes. The results showed a differential distribution of the mRNAs for these two receptor forms in the rat. The mRNA for CRF2 alpha is found almost exclusively in the brain, particularly in the hypothalamus, lateral septum, and olfactory bulb, whereas the mRNA for CRF2 beta appears to be both in the brain and in the periphery, with the greatest abundance in the heart and skeletal muscle. Thus, the data suggest that these alternatively spliced forms of the CRF2 receptor may represent functionally distinct CRF receptors. In addition, it highlights the importance of probe specificity for in situ hybridization studies.