The C family G-protein-coupled receptors contain members that sense amino acid and extracellular cations, of which calcium-sensing receptor (CASR) is the prototypic extracellular calcium-sensing receptor. Some cells, such as osteoblasts in bone, retain responsiveness to extracellular calcium in CASR-deficient mice, consistent with the existence of another calcium-sensing receptor. We examined the calcium-sensing properties of GPRC6A, a newly identified member of this family. Alignment of GPRC6A with CASR revealed conservation of both calcium and calcimimetic binding sites. In addition, calcium, magnesium, strontium, aluminum, gadolinium, and the calcimimetic NPS 568 resulted in a dose-dependent stimulation of GPRC6A overexpressed in human embryonic kidney cells 293 cells. Also, osteocalcin, a calcium-binding protein highly expressed in bone, dose-dependently stimulated GPRC6A activity in the presence of calcium but inhibited the calcium-dependent activation of CASR. Coexpression of ␤-arrestins 1 and 2, regulators of G-protein signaling RGS2 or RGS4, the RhoA inhibitor C3 toxin, the dominant negative G␣ q -(305-359) minigene, and pretreatment with pertussis toxin inhibited activation of GPRC6A by extracellular cations. Reverse transcription-PCR analyses showed that mouse GPRC6A is widely expressed in mouse tissues, including bone, calvaria, and the osteoblastic cell line MC3T3-E1. These data suggest that in addition to sensing amino acids, GPRC6A is a cation-, calcimimetic-, and osteocalcin-sensing receptor and a candidate for mediating extracellular calcium-sensing responses in osteoblasts and possibly other tissues.

BackgroundGPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown.Methods/Principal FindingsIn this study, we created and characterized the phenotype of GPRC6A −/− mice. We observed complex metabolic abnormalities in GPRC6A −/− mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A −/− mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A −/− mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A−/− mice exhibited increments in urine Ca/Cr and PO4/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A −/− mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone.Conclusions/Significance GPRC6A−/− mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.

Dorsal-ventral patterning within the ectoderm of the Drosophila embryo requires seven zygotic genes, including short gastrulation (sog). Here we demonstrate that sog, which is expressed in the ventrolateral region of the embryo that gives rise to the nerve cord, is functionally homologous to the chordin gene of Xenopus, which is expressed in the dorsal blastopore lip of the embryo and in dorsal mesoderm, in particular the notochord. We show by injections of messenger RNA that both sog and chordin can promote ventral development in Drosophila, and that sog, like chordin, can promote dorsal development in Xenopus. In Drosophila, sog antagonizes the dorsalizing effects of decapentaplegic (dpp), a member of the transforming growth factor-beta family. One of the dpp homologues in vertebrates, bmp-4, is expressed ventrally in Xenopus and promotes ventral development. We show that dpp can promote ventral fates in Xenopus, and that injection of sog mRNA counteracts the ventralizing effects of dpp. These results suggest the molecular conservation of dorsoventral patterning mechanisms during evolution.

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Patterns of decapentaplegic (dpp) transcripts derived from the intact gene were compared to the patterns of transcripts generated by partial dpp transgenes in Drosophila embryos. Sequences closest to the dpp coding regions, the dpp hin region, were sufficient to express ZacZ-tagged mRNA in patterns indistinguishable from the patterns of endogenous dpp expression in the dorsal and terminal cells at the blastoderm stage, in the dorsal ectoderm during germ band elongation, and in narrow stripes of ectodermal cells along the dorsal edge of the ectoderm and at the boundary between the lateral and ventral neurogenic regions during germ band shortening. The latter pattern of expression responded to the segment polarity genes naked and wingless. However, these dpp sequences were not sufficient to drive ZucZ-tagged mRNA expression in other cells normally expressing dpp, including cells in the gnathal segments, the clypeolabrum, the foregut, the midgut visceral mesoderm, and the hindgut. Two separate regulatory regions were found in the dpp hin region. A 479 bp region upstream of the promoter was necessary for the segmented pattern of expression in the lateral ectoderm and for expression in the midgut endoderm. Cis-acting elements in the 2 kbp second intron directed expression in the dorsal and terminal regions of the blastoderm, acted on a heterologous promoter, the P-element promoter, and responded to pattern information derived from the maternal effect dorsal/ventral patterning genes. 0 1994 Wiley-Liss, Inc.

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.