Expression of adenylate cyclase-coupled osseous parathyroid hormone and parathyroid hormone-like peptide receptors in Xenopus oocytes.
Functional parathyroid hormone (PTH) and PTH-like peptide receptors were expressed in Xenopus laevis oocytes after injection of poly(A)+ RNA isolated from the rat osteogenic sarcoma cell line, UMR 106. Increases in cAMP were seen in individual oocytes in response to added bovine (b) PTH-(1-34) (10(-6) M), human (h) PLP-(1-34) (hPLP-(1-34), 10(-6) M), isoproterenol (10(-4) M), and forskolin (10(-4) M). Although both intracellular and extracellular cAMP levels were stimulated approximately 1.5-2-fold by these agonists, intracellular concentrations of cAMP were substantially higher than extracellular concentrations. Peak increases with bPTH-(1-34) occurred after a 30-min incubation with the hormone 48 h after oocyte injection. bPTH-(1-34) caused a concentration-dependent augmentation of cAMP in injected oocytes, and the in vitro antagonist hPLP-(3-34) produced dose-dependent inhibition of both bPTH-(1-34)- and hPLP-(1-34)-stimulated cAMP accumulation. Specific binding of PTH to oocyte membranes was also demonstrated 48 h after oocyte injection with UMR 106 cell mRNA. Following size fractionation of isolated UMR 106 poly(A)+ RNA by sucrose density gradients, mRNA directing the expression of both PTH- and PLP-stimulated cAMP in oocytes appeared in the 3.5-4.9-kilobase fraction. These results demonstrate that adenylate cyclase-coupled osseous PTH and PLP receptors can be expressed after injection of naturally occurring mRNA into Xenopus oocytes, that PTH- and PLP-stimulated increases in cAMP concentrations can be detected in individual oocytes injected with bone cell-derived mRNA, that PTH and PLP appear to cross-react at a common receptor after injection of UMR 106 cell mRNA into oocytes, and that size selection of mRNA encoding the PTH and PLP receptors can be achieved by density gradient centrifugation. These studies, therefore, indicate the potential usefulness of the Xenopus oocyte system in expression cloning of PTH and PLP receptor cDNAs and illustrate the feasibility of employing this system to examine the biology of PTH and PLP receptors.
Horiuchi
T
T
Calcium Research Laboratory, McGill University, Montreal, Quebec, Canada.
Champigny
C
C
Rabbani
S A
SA
Hendy
G N
GN
Goltzman
D
D
D000262:Adenylyl Cyclases / Q000235:genetics*; D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D008024:Ligands; D012516:Osteosarcoma / Q000378:metabolism; D010281:Parathyroid Hormone / Q000235:genetics* / Q000494:pharmacology; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology; D044168:Receptor, Parathyroid Hormone, Type 1; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D018016:Receptors, Parathyroid Hormone; D014407:Tumor Cells, Cultured; D014981:Xenopus
D008024:Ligands; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; C054296:parathyroid hormone-related protein (1-34); C025076:parathyroid hormone (1-34), bovine; D000242:Cyclic AMP; D000262:Adenylyl Cyclases