Structure and functional expression of a complementary DNA for porcine parathyroid hormone /parathyroid hormone-related peptide receptor

Abstract

A complementary DNA (cDNA) encoding the receptor for porcine parathyroid hormone /parathyroid hormone-related peptide (PTH/PTHrP) was isolated from a porcine kidney cDNA library. The porcine PTH/PTHrP receptor is a 585 amino acid protein containing seven putative membrane-spanning domains. The porcine PTH/PTHrP receptor has amino acid identity of 95.6%, 80.4%, and 88.7% with human, opossum, and rat PTH/PTHrP receptors, respectively and 53.4% identity to the recently cloned human PTH2 receptor. The receptor cDNA was subsequently cloned into a mammalian cell expression vector (pRC/CMV) which contains a human cytomegalovirus promoter. A human kidney cell line (293), stably transfected with this vector, expressed the receptor at a high level and, when challenged with human PTH(1–34), increased cytoplasmic CAMP and inositol triphosphate production. Radioligand binding studies revealed that the receptor bound both human PTH(1–34), and PTHM(1–36). Scatchard analyses of three clones showed that the cells harbor a single class of high affinity receptor (K d= 1−4 nM for human PTH(1–34)) but had varying receptor numbers (10) 5– 10 6 receptors/cell). In contrast to PTH(1–34), the [Arg 2]PTH(1–34) analog bound to the porcine PTH/PTHrP receptor with low affinity and was a weak agonist for cAMP stimulation with the cloned receptor. These response characteristics differentiate the porcine receptor from the previously cloned rat and opossum PTH/PTHrP receptors.