Relaxin is a hormone important for the growth and remodeling of reproductive and other tissues during pregnancy. Although binding sites for relaxin are widely distributed, the nature of its receptor has been elusive. Here, we demonstrate that two orphan heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors, LGR7 and LGR8, are capable of mediating the action of relaxin through an adenosine 3',5'-monophosphate (cAMP)-dependent pathway distinct from that of the structurally related insulin and insulin-like growth factor family ligand. Treatment of antepartum mice with the soluble ligand-binding region of LGR7 caused parturition delay. The wide and divergent distribution of the two relaxin receptors implicates their roles in reproductive, brain, renal, cardiovascular, and other functions.

Adaptive stress responses mediated by the endocrine, autonomic, cardiovascular and immune systems are essential for the survival of the individual. Initial stress-induced responses provide a vital short-term metabolic lift, but prolonged or inappropriate exposure to stress can compromise homeostasis thereby leading to disease. This 'fight-or-flight' response is characterized by the activation of the corticotropin-releasing hormone (CRH)-adrenocorticotropin-glucocorticoid axis, mediated by the type 1 CRH receptor. In contrast, the type 2 CRH receptor mediates the stress-coping responses during the recovery phase of stress. We identified human stresscopin (SCP) and stresscopin-related peptide (SRP) as specific ligands for the type 2 CRH receptor. The genes encoding these peptides were expressed in diverse peripheral tissues as well as in the central nervous system. Treatment with SCP or SRP suppressed food intake, delayed gastric emptying and decreased heat-induced edema. Thus SCP and SRP might represent endogenous ligands for maintaining homeostasis after stress, and could allow the design of drugs to ameliorate stress-related diseases.

Calcitonin, calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and amylin belong to a unique group of peptide hormones important for homeostasis in diverse tissues. Calcitonin is essential for calcium balance, whereas CGRP and ADM are important for neurotransmission and cardiovascular and respiratory regulation. Based on phylogenetic analysis, we identified intermedin as a novel member of the calcitonin/CGRP peptide family. Analysis of intermedin expression indicated that intermedin is expressed primarily in the pituitary and gastrointestinal tract. Intermedin increased cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and competed with labeled CGRP for binding to its receptors in these cells. In addition, treatment of 293T cells expressing recombinant calcitonin receptor-like receptor (CRLR) and one of the three receptor activity-modifying proteins (RAMPs) showed that a CRLR/RAMP receptor complex is required for intermedin signaling. In contrast to CGRP and ADM, which exhibited a preferential stimulation of CRLR when co-expressed with RAMP1 and RAMP2 or RAMP3, respectively, intermedin represents a nonselective agonist for the RAMP coreceptors. In vivo studies demonstrated that intermedin treatment led to blood pressure reduction in both normal and spontaneously hypertensive rats via interactions with the CRLR/RAMP receptor complexes. Furthermore, in vivo treatment in mice with intermedin led to suppression of gastric emptying activity and food intake. Thus, identification of intermedin as a novel member of the calcitonin/CGRP peptide family capable of signaling through CRLR/ RAMP receptor complexes provides an additional player in the regulation of peripheral tissues by CRLR and will allow development of new therapeutic agents for pathologies associated with diverse vascular and gastrointestinal disorders.Originally isolated as a polypeptide hormone essential for calcium balance (1, 2), calcitonin belongs to a group of peptide hormones including ␣-CGRP, 1 ␤-CGRP, adrenomedullin (ADM), and amylin (3). Among these tissue-specific peptides, ADM and CGRP are important endocrine and neurocrine integrators of homeostasis in the vascular and respiratory systems, whereas amylin is essential for optimal glucose metabolism. The biological actions of these peptides are mediated via binding to two closely related type II G protein-coupled receptors (GPCRs), the calcitonin receptor, and the calcitonin receptorlike receptor (CRLR) (4, 5). Although the calcitonin receptor is the main mediator for calcitonin action, it also binds amylin. Recent cloning and functional studies have shown that CGRP, ADM, and, to a lesser extent, amylin interact with different combinations of CRLR and the three receptor activity-modifying proteins (RAMPs) (5, 6). Studies using mutant mice deficient in ␣-CGRP, ADM, or amylin have indicated that CRLR could be important for cardiovascular morphogenesis, sensory neurotransmission, inflammatory reactions, nociceptive behavior, and glucose homeostasis (7-12). Thus, ...

In the intracellular death program, heteroand homodimerization of different anti-and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bf l-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviralderived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

Several orphan G protein-coupled receptors homologous to gonadotropin and thyrotropin receptors have recently been identified and named as LGR4 -8. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a relaxin family member expressed in testisDuring fetal development, the sexual dimorphic position of the gonads in mammals is dependent on the differential development of two ligaments. In males, growth of the gubernaculum and regression of the cranial suspensory ligament results in transabdominal descent of the testes. Impaired testicular descent (cryptorchidism) is a prevalent congenital abnormality in humans, found in 2% of male births. INSL3, 1 also known as Leydig insulin-like peptide or relaxin-like factor, is one of the seven relaxin-like genes in humans known to be expressed in Leydig cells of fetal and adult testes as well as in theca and luteal cells of the postnatal ovary (1). Male mice mutant for INSL3 exhibit bilateral abdominal cryptorchidism (2, 3) whereas female mice overexpressing INSL3 showed ovary descent and displayed bilateral inguinal hernia (4). Although INSL3 binds to gubernacular homogenates (5, 6) and induces growth of rat gubernaculum in whole organ cultures (7), the exact nature of the INSL3 receptor is unknown.A recent study indicated that transgene integration in crsp mice resulted in a 550-kb deletion located upstream of the Brca2 gene, leading to defective testis descent. Because a candidate gene encoding a G protein-coupled receptor homologous to human LGR8 was deleted in these mice (8), we tested whether INSL3 is the cognate ligand for LGR8 based on the observed common phenotypes of potential ligand-receptor pairs in null mice. Here, we report that INSL3 is capable of binding LGR8, leading to the stimulation of cAMP production and thymidine incorporation in the gubernaculum. EXPERIMENTAL PROCEDURESOvine and rat INSL3 were chemically synthesized and characterized as described (7, I-Streptavidin and streptavidin conjugated to horseradish peroxidase (HRP) were purchased from Amersham Biosciences, whereas foskolin, glucagon, collagenase, and trypsin were from Sigma. Sprague-Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). Animals were anesthetized and killed using CO 2 . Animal care was consistent with institutional and NIH guidelines.Human 293T cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 100 g/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. When 70 -80% confluent, cells were transfected with 10 g of plasmid using the calcium phosphate precipitation method (10). After 18 -24 h of incubation, media were replaced with DMEM/F12 containing 10% FBS. Forty-eight hours after transfection, cells (10 5 /ml) were preincubated at 37°C for 30 min in the presence of 0.25 mM 3-isobutyl-1-methylxanthine (IBMX) before treatment with or without hormones for 12 h. Total cAMP content was measured in triplicate by a specific radioimmunoassay (11). All experiments we...