Physiologic responsiveness to parathyroid hormone (PTH), the major regulator of bone turnover and extracellular calcium homeostasis, is determined, not only by the plasma concentration of PTH, but also by the number of functional receptors in target tissues. PTH-related peptide (PTHrP), which causes hypercalcemia in cancer patients and is thought to have paracrine functions in a number of tissues and during fetal development, binds to the same PTH receptor. Regulation of transcription of the PTH/PTHrP receptor gene is likely to determine which tissues express these receptors and how many receptors are expressed in these tissues. The goal of this research is to determine how tissue specificity and extent of receptor expression are controlled in the two classic PTH target tissues, bone and kidney. cDNA clones encoding a common PTH/PTHrP receptor from opossum kidney and rat osteosarcoma cells have been recently isolated. The rat cDNA was then used as a probe to isolate a rat genomic clone. Partial characterization of this clone indicates the presence of multiple intervening sequences.
Specific Aim 1 will involve characterization of this clone and isolation of other overlapping clones to determine the structure of the PTH/PTHrP receptor gene. The molecular mechanisms regulating expression of the PTH receptor during the differentiation of the pluripotent mesenchymal C3H1OT1/2 cells into bone-forming osteoblasts (Specific Aim 2) and in response to important hormonal regulators in well-differentiated bone and kidney cells (Specific Aim 3) will be determined.
Specific Aim 4 will involve the determination of the DNA sequences responsible for tissue-specific expression and hormonal regulation of the rat PTH/PTHrP receptor gene. These regulatory DNA sequences are likely to interact with specific regulatory proteins. Therefore, the interaction of the regulatory sequences with nuclear proteins prepared from relevant cells will be studied (Specific Aim 5). Characterization of specific protein-DNA interaction provide a molecular model for understanding the tissue-specific expression and regulation of the PTH receptor gene. The goal of Specific Aim 6 is to clone cDNA(s) encoding regulatory proteins that bind to and activate specific DNA sequences responsible for cell-specific expression and downregulation by PTH of the PTH receptor gene. Achievement of the goals of this grant will provide an understanding of how the PTH receptor is expressed in bone and kidney cells and how the expression of this receptor is regulated.
