| Parathyroid hormone (PTH) precisely regulates serum calcium concentration by acting on calcium stores in bone, and modifying renal calcium reabsorption and vitamin D biosynthesis. PTH exerts its actions on, bone and kidney via a G-protein-coupled seven- transmembrane receptor (PTH1 Rc), of the glucagon/vasoactive intestinal peptide/secretin receptor subfamily, which is able to signal via both cAMP and IP3/intracellular calcium 2nd messenger pathways.;Very little is known at the molecular level regarding the binding surface epitopes of the human PTH1 receptor or the actual amino acid-to-amino acid contact points at the interface of hormone and receptor. Furthermore, while the second messenger pathways have been well characterized, the temporal pattern of gene expression and other early signaling events following PTH1 Rc stimulation are not well understood.;I have employed the methodology of photoaffinity crosslinking as an approach to studying the amino acid-to-amino acid contact points between PTH and its receptor. Following cloning and biologic characterization, the recombinant human (h) PTH1 Rc was overexpressed in a human cell line. Crosslinking of these hPTH1 Rc-expressing cells to a PTH ligand containing a photoaffinity moiety at position 13 revealed a specific, competeable ∼80–90kDa protein conjugate. Chemical and enzymatic cleavage of this hormone-receptor conjugate, followed by gel electrophoresis allowed our laboratory to identify a hormone-Rc contact domain: position 13 of PTH(1-34) interacts with a region of the PTH1 Rc bordered by residues 173–189. Using a combination of site-directed mutagenesis and biochemical analyses, this contact domain was further refined to residues 182–189, and residue Arg 186 was found to be critical for the crosslinking of Lys 13-substituted PTH(1-34).;A PTH ligand containing the photoaffinity moiety at position one of PTH crosslinks to a region of the hPTH1 Rc spanning portions of the sixth transmembrane domain and the third extracellular loop (409–437). Previous studies, in our laboratory, predicted a methionine in this region as the contact point. Evidence from site-directed mutagenesis of this residue further supports the implication of M425 as the contact point of position one of PTH.;In addition, in order to better understand the changes in gene expression which occur following stimulation of bone cells by PTH, we used differential mRNA display to compare PTH-treated and untreated Saos-2/B10 cells (an osteoblast-like cell line). This study identified a number of PTH-regulated genes, including a novel PTH-responsive gene. This novel mRNA was maximally down-regulated within one hour of PTH(1-34) stimulation. The mRNA appears to be the product of a single gene, which is alternatively spliced to produce multiple transcripts in all tissues, except bone and bone-derived cells, in which only one major transcript is apparent. |
