Coupling of the PTH/PTHrP receptor to multiple G-proteins. Direct demonstration of receptor activation of Gs, Gq/11, and Gi(1) by [alpha-32P]GTP-gamma-azidoanilide photoaffinity labeling.
Parathyroid hormone (PTH) elicits many of its physiological effects by activating distinct, G-protein-coupled signaling cascades that lead to synthesis of cyclic AMP and hydrolysis of phosphatidylinositol 4,5-bisphosphate. Using the nonhydrolyzable photo-reactive GTP analog [alpha-32P]GTP-gamma-azidoanilide (GTP-AA) and peptide antisera raised against G-protein alpha-subunits, we studied coupling of the PTH receptor to G-proteins in rat osteoblast-like cells (ROS 17/2.8), and in human embryonal kidney cells expressing the cloned human PTH/parathyroid hormone-related peptide (PTHrP) receptor at 40,000 receptors/cell (C20) or 400,000 receptors/cell (C21). Incubation of C21 membranes (but not C20 membranes) with [Nle8,18, Tyr34]-bovine PTH(1-34) amide (bPTH[1-34]) led to concentration-dependent incorporation of GTP-AA into the two isoforms of G alpha s, into G alpha q/11, and to a much lesser extent into G alpha i(1). In ROS 17/2.8 cells, bPTH(1-34) increased the incorporation of GTP-AA into G alpha s, but not into G alpha q/11 or G alpha i. The ability of bPTH(1-34) to increase labeling of G alpha s and G alpha q/11 was correlated with the receptor-dependent sensitivity of the adenylyl cyclase and phospholipase C signaling pathways to the hormone.
Division of Endocrinology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. wschwind@welchlink.welch.jhu.edu
W F
WF
Schwindinger
J
J
Fredericks
L
L
Watkins
H
H
Robinson
J M
JM
Bathon
M
M
Pines
L J
LJ
Suva
M A
MA
Levine
D000262:Adenylyl Cyclases / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004622:Embryo, Mammalian; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007668:Kidney; D010006:Osteoblasts / Q000378:metabolism; D012516:Osteosarcoma; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases