Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation.
Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.
Department of Medicine, Howard Hughes Medical Institute, Duke University, Medical Center, Durham, North Carolina 27710, USA.
Diane
D
Gesty-Palmer
Minyong
M
Chen
Eric
E
Reiter
Seungkirl
S
Ahn
Christopher D
CD
Nelson
Shuntai
S
Wang
Allen E
AE
Eckhardt
Conrad L
CL
Cowan
Robert F
RF
Spurney
Louis M
LM
Luttrell
Robert J
RJ
Lefkowitz
D019390:Arrestins / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008024:Ligands; D020935:MAP Kinase Signaling System; D008301:Maleimides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D007211:Indoles; D007546:Isoquinolines; D008024:Ligands; D008301:Maleimides; D034741:RNA, Small Interfering; D044168:Receptor, Parathyroid Hormone, Type 1; D013449:Sulfonamides; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins; C070515:bisindolylmaleimide I; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide