Molecular cloning of the gene encoding the mouse parathyroid hormone/parathyroid hormone-related peptide receptor.

Abstract

The parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) is a G-protein-coupled receptor containin seven predicted transmembrane do . We have isolated and characterized recombinant bacteriophage AEMBL3 genomic clones containing the mouse PTHR gene, including 10 kilobases of the promoter region. The gene spans >32 kilobases and in divided into 15 exons, 8 of which contain the tansmembrne dans. The PTHR exons contining the predicted membrane-snning d are beterogeneous in length and three of the exon-ntron boundaries fall within putative transmembrane sequences, suggestin that the exons did not arise from duplication events. This arrangement is closely related to that of the growth hormone releasing factor receptor gene, particularly in the transmembrane region, providing strong evidence that the two genes evolved from a common precursor. Transcription is initiated principally at a series of sites over a 15-base-pair region. The proximal promoter region is highly (G+C)-rich and lacks an apparent TATA box or initiator element homologies but does contain CCGCCC motifs. The presumptive amino acid sequence of the encoded receptor is 99%, 91%, and 76% identical to those of the rat, human, and opossum receptors, respectively. There is no consensus polyadenylylation signal in the 3' untnted region. The poly(A) tail ofthe PTHR transcript begins 32 bases downstream ofa 35-base-long A-rich sequence, suggesting that this region directs polyadenylylation.The parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) is bound specifically by a conserved 34-amino acid region present in both parathyroid hormone (PTH) and PTH-related peptide (PTHrP). PTH regulates calcium and phosphate metabolism by binding to receptors expressed in kidney and bone (1-5). PTHrP was first identified as a major cause of malignancy-associated hypercalcemia (6, 7); however, its normal physiological role remains largely unknown. Whereas PTH expression is limited to the parathyroid, PTHrP is expressed in a wide variety of normal and malignant tissues and appears to act mainly in a para-or autocrine manner (8-11). The PTHR is a G-protein-coupled receptor containing seven predicted transmembrane domains (refs. 1-3 and references therein). Binding of ligand to the PTHR stimulates cAMP production, raises intracellular calcium, and increases levels of inositol 1,4,5-trisphosphate (2).The G-protein-coupled family of receptors is vast and includes receptors for peptide hormones, >100 odorants, neurotransmitters, and a number of other regulatory factors (12). Based on similarities between ligands and receptors (13, 14), the PTHR belongs to a subfamily that includes receptors for growth hormone releasing factor, vasoactive intestinal peptide, calcitonin, secretin, glucagon-like peptide, and glucagon. Genes for several mammalian adrenergic and serotonin receptors have been cloned and are intronless (15)(16)(17)(18)(19). Although the luteinizing hormone receptor contains 11 exons, the transmembrane and cytoplasmic regions of...

Sequencing of eleven introns in genomic DNA encoding rat glucagon receptor and multiple alternative splicing of its mRNA

“…The sequence and the size of this GH-RH receptor gene introns was not reported. Recently, the entire sequence of mouse PTH receptor gene was reported [30]. The coding sequence spans 35 kb and is interrupted by 12 introns, at positions similar to the GRF and glucagon receptor gene introns, suggesting that these genes diverged from a common ancestor [30].…”

Effect of Bone Morphogenetic Protein-2 (BMP-2) or Troglitazone, as an Inducer of Osteogenic Cells or Adipocytes, on Differentiation of a Bone Marrow Mesenchymal Progenitor Cell Line Established from Temperature-Sensitive (ts) Simian Virus (SV) 40 T-Antigen Gene Transgenic Mice

“…28) Briefly, cDNA was synthesized using oligodT primers and Moloney murine leukemia virus reverse transcriptase, followed by PCR amplification with the appropriate gene primers specific for osteogenic cell differentiation markers, such as core-binding factor a -1 (Cbfa1), alkaline phosphatase (ALP), type I collagen (Col), bone Gla-protein (BGP), osteopontin (OPN), parathyroid hormone receptor (PTHR) and vitamin D receptor (VDR), adipocyte differentiation marker, peroxisome proliferator-activated receptor g (PPARg), adipsin (ADPS), and lipoprotein lipase (LPL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) produced from the reported cDNA sequences, respectively. [29][30][31][32][33][34][35][36][37][38][39] The oligonucleotide primers were synthesized by Oligo (Tsukuba, Japan). The primers used were as follows: Cbfa1 forward 21-mer, 5Ј-CAGTTCCCAAGCATTTCATCC-3Ј; Cbfa1 reverse 21-mer, 5Ј-CAGCGTCAACACCATCATTCT-3Ј; ALP forward 20-mer, 5Ј-GTGGATACACCCCCCGGG-GC-3Ј; ALP reverse 22-mer, 5Ј-GGTCAAGGTTGGCCC-CAATGCA-3Ј; Col forward 21-mer, 5Ј-TGGCTATGAC-TTTGGTTTTGA-3Ј; Col reverse 21-mer, 5Ј-GTTCTTC-GCTGGGGTGTTTAC-3Ј; BGP forward 21-mer, 5Ј-CCTG-GCTGCGCTCTGTCTCTC-3Ј; BGP reverse 21-mer, 5Ј-TTATTTTGGAGCTGCTGTGAC-3Ј; OPN forward 21-mer, 5Ј-CACCATTCGGATGAGTCTGAT-3Ј; OPN reverse 21-mer, 5Ј-GCTGCCCTTTCCGTTGTTGTC-3Ј; PTHR forward 21-mer, 5Ј-CATGTCTCTTGCCTCCCTCAC-3Ј; PTHR reverse 21-mer, 5Ј-TAAGTACAGTCCCTCCACCAG-3Ј; VDR forward 21-mer, 5Ј-TGGCAGCCAAGACTACAAATA-3Ј; VDR reverse 21-mer, 5Ј-CGGCTGGAAGGAGAGGGAA-CG-3Ј; PPARg forward 21-mer, 5Ј-GCTCCAAGAATACC-AAAGTGC-3Ј; PPARg reverse 21-mer, 5Ј-GTCGATCCCG-CCCAAACCTGA-3Ј; ADPS forward 21-mer, 5Ј-CTATCC-CAGAATGCCTCGTTG-3Ј; ADPS reverse 21-mer, 5Ј-CTT-TTTGCCATTGCCACAGAC-3Ј; LPL forward 21-mer, 5Ј-GCCAAGAGAAGCAGCAAGATG-3Ј; LPL reverse 21-mer, 5Ј-CCCTAGCACAGAAGATGACCT-3Ј; GAPDH forward 20-mer, 5Ј-ACCACAGTCCATGCCATCAC-3Ј; GAPDH reverse 20-mer, 5Ј-TCCACCACCCTGTTGCTGTA-3Ј.…”

Vitamin D3 differentially regulates parathyroid hormone/parathyroid hormone-related peptide receptor expression in bone and cartilage

“…The PTHR is widely expressed, with the highest levels being found in kidney and bone (33). The mouse gene encoding the PTHR is composed of at least 17 exons, and its expression is regulated by at least two promoters (34,35), which give rise to two transcripts that differ in their 5′ untranslated sequences, but not in their coding regions. Transcripts from the upstream mouse promoter (P1) contain sequences derived from two 5′ untranslated region exons, U1 and U2, and are highly expressed in kidney and weakly expressed in liver.…”