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, ...

Glycoprotein hormone receptors, including LH receptor, FSH receptor, and TSH receptor, belong to the large G protein-coupled receptor (GPCR) superfamily but are unique in having a large ectodomain important for ligand binding. In addition to two recently isolated mammalian LGRs (leucine-rich repeat-containing, G protein-coupled receptors), LGR4 and LGR5, we further identified two new paralogs, LGR6 and LGR7, for glycoprotein hormone receptors. Phylogenetic analysis showed that there are three LGR subgroups: the known glycoprotein hormone receptors; LGR4 to 6; and a third subgroup represented by LGR7. LGR6 has a subgroup-specific hinge region after leucine-rich repeats whereas LGR7, like snail LGR, contains a low density lipoprotein (LDL) receptor cysteine-rich motif at the N terminus. Similar to LGR4 and LGR5, LGR6 and LGR7 mRNAs are expressed in multiple tissues. Although the putative ligands for LGR6 and LGR7 are unknown, studies on single amino acid mutants of LGR7, with a design based on known LH and TSH receptor gain-of-function mutations, indicated that the action of LGR7 is likely mediated by the protein kinase A but not the phospholipase C pathway. Thus, mutagenesis of conserved residues to allow constitutive receptor activation is a novel approach for the characterization of signaling pathways of selective orphan GPCRs. The present study also defines the existence of three subclasses of leucine-rich repeat-containing, G protein-coupled receptors in the human genome and allows future studies on the physiological importance of this expanding subgroup of GPCR.

Human thyrotropin (TSH), luteotropin (LH), follitropin (FSH), and chorionic gonadotropin are members of the heterodimeric glycoprotein hormone family. The common alpha subunit forms noncovalent heterodimers with different beta subunits. Two novel human glycoprotein hormonelike genes, alpha2 (A2) and beta5 (B5), recently have been identified. Using a yeast two-hybrid assay, the two subunits were found as potential heterodimerization partners. Immunological analyses confirmed the heterodimerization of A2 and B5 in transfected cells and their colocalization in the anterior pituitary. Recombinant A2/B5 heterodimeric glycoproteins, purified using cation exchange and size fractionation chromatography, activated human TSH receptors, but not LH and FSH receptors, and showed high affinity to TSH receptors in a radioligand receptor assay. The heterodimer also stimulated cAMP production and thymidine incorporation by cultured thyroid cells and increased serum thyroxine levels in TSH-suppressed rats in vivo. This new heterodimeric glycoprotein hormone was named as thyrostimulin based on its thyroid-stimulating activity. The expression of thyrostimulin in the anterior pituitary known to express TSH receptors suggested a paracrine mechanism. The present discovery of a new ligand based on genomic approaches could facilitate the understanding of the physiological roles of extra-thyroid TSH receptor systems and the structural-functional basis of receptor signaling by related glycoprotein hormones.

The canonical members of the human glycoprotein hormone subunit family of cystine knot-forming polypeptides include the common alpha-subunit, and four beta-subunit genes, FSHbeta, LHbeta, TSHbeta, and hCGbeta. Using pairwise sequence analysis of the complete human genome, we have identified two novel glycoprotein hormone subunit-related genes. Based on unique sequence similarity to the alpha- and beta-subunits of glycoprotein hormones, they were named glycoprotein-alpha2 (GPA2) and glycoprotein-beta5 (GPB5), respectively. PCR analysis using a panel of human cDNAs from 14 different tissues demonstrated that GPB5 is similar to other beta-subunits showing restricted tissue expression, mainly in pituitary and brain. In contrast, the GPA2 transcript is found in diverse tissues. Furthermore, immunoreactive GPA2 and GPB5 were detected in the anterior pituitary of mouse and frog, whereas the expression of GPA2 and GPB5 in transfected cells resulted in the secretion of recombinant polypeptides in conditioned medium. After GenBank searches in lower organisms, glycoprotein hormone beta-subunit-related genes were identified from the genome of nematode Caenorhabditis elegans, hookworm Ancylostoma caninum, and Drosophila melanogaster. The evolutionary conservation of these invertebrate homologs can be seen in several key sequence characteristics, and the data suggest that the glycoprotein hormone beta-subunit gene ancestor evolved before the emergence of bilateral metazoa, thus providing a better understanding of the evolution of this group of classic polypeptide hormones and their receptors. Studies of the complete inventory of genes homologous to glycoprotein hormone subunits in the human genome and lower organisms will allow future functional characterization and identification of their respective receptors.

IntroductionHuman thyrotropin (TSH), follitropin (FSH), lutropin (LH), and chorionic gonadotropin (CG) are members of the glycoprotein hormone family derived from heterodimerization of a common α subunit with hormone-specific β subunits. These hormones were originally purified from the anterior pituitary (TSH, LH, and FSH) and placenta (human CG) and shown to activate specific G protein-coupled receptors in the thyroid (TSH receptor) and gonads (LH and FSH receptors), respectively (1-4). These three pituitary-derived glycoprotein hormones form the basis of the classic pituitary-peripheral target feedback systems and are essential for the development and differentiation of thyroid and gonadal tissues. In particular, TSH is essential for the production of iodothyronines by the thyroid gland and disorders of the pituitary-thyroid gland-thyroid hormone axis lead to disturbances of essentially all metabolic pathways and organs (5, 6).Based on GenBank searches, we identified two additional human glycoprotein hormone subunitlike genes and named them α2 (A2) and β5 (B5), due to their structural similarities to known subunits and the chronology of discovery (7). (The GenBank accession numbers for A2 and B5 are AF403384 and AF403430, respectively.) A2 and B5 have conserved cysteine residues, similar to those found in the well-characterized α and β subunits (8, 9) important for the formation of key disulfide bonds. Like all other glycoprotein hormone subunits, A2 and B5 have the unique cystine knot structure characteristic of proteins related to the TGF-β, the PDGF, and the bone morphogenetic protein families (10, 11). Because the putative A2 subunit is likely to combine with either known or novel β subunits to yield bioactive heterodimeric hormones, we performed a yeast two-hybrid protein-protein interaction screen to identify potential dimerization partners for A2 and found interactions between A2 and B5. We generated Ab's against A2 and B5 to confirm the interactions between these putative subunits and their colocalization in the anterior pituitary. Following testing for the activation of glycoprotein hormone receptors, Human thyrotropin (TSH), luteotropin (LH), follitropin (FSH), and chorionic gonadotropin are members of the heterodimeric glycoprotein hormone family. The common α subunit forms noncovalent heterodimers with different β subunits. Two novel human glycoprotein hormonelike genes, α2 (A2) and β5 (B5), recently have been identified. Using a yeast two-hybrid assay, the two subunits were found as potential heterodimerization partners. Immunological analyses confirmed the heterodimerization of A2 and B5 in transfected cells and their colocalization in the anterior pituitary. Recombinant A2/B5 heterodimeric glycoproteins, purified using cation exchange and size fractionation chromatography, activated human TSH receptors, but not LH and FSH receptors, and showed high affinity to TSH receptors in a radioligand receptor assay. The heterodimer also stimulated cAMP production and thymidine incorporation by cultured th...