title: Calcium-sensing receptor-mediated ERK1/2 activation requires Galphai2 coupling and dynamin-independent receptor internalization.
abstract: The calcium-sensing receptor (CaR) recently has been shown to activate MAP kinase (ERK1/2) in various cell types as well as in heterologous expression systems. In this study we show that the CaR agonist NPS R-467 (1 microm), which does not activate the CaR by itself, robustly activates ERK1/2 in the presence of a low concentration of Ca(2+) (0.5 mm CaCl(2)) in human embryonic kidney (HEK) cells permanently expressing the human CaR (HEK-hCaR). Ca(2+) (4 mm) also activates ERK1/2 but with differing kinetics. CaR-dependent ERK1/2 activation begins to desensitize to 4 mm Ca(2+) after 10 min, whereas there is no desensitization to NPS R-467/CaCl(2) as late as 4 h. Moreover, recovery from desensitization occurs as rapidly as 30 min with 4 mm CaCl(2). Pretreatment of HEK-hCaR cells with concanavalin A (250 microg/ml) to block CaR internalization completely eliminated the NPS R-467/CaCl(2)-mediated ERK1/2 activation but did not block the 2-min time point of 4 mm Ca(2+)-mediated ERK1/2 activation. Neither dominant-negative dynamin (K44A) nor dominant-negative beta-arrestin inhibited ERK1/2 activation by either CaR agonist treatment, suggesting that CaR-elicited ERK1/2 signaling occurs via a dynamin-independent pathway. Pertussis toxin pretreatment partially attenuated the 4 mm Ca(2+)-ERK1/2 activation; this attenuated activity was completely restored by co-expression of the Galpha(i2) (C351I) but not Galpha(i1) (C351I) or Galpha(i3) (C351I) G proteins, PTX-insensitive G protein mutants. Taken together, these data suggest that both 4 mm Ca(2+) and NPS R-467/CaCl(2) activate ERK1/2 via distinguishable pathways in HEK-hCaR cells and may represent a nexus to differentially regulate differentiation versus proliferation via CaR activation.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, 78229-3900, USA.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Holstein
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Berg
authors.forename: L M Fredrik
authors.initials: LM
authors.lastname: Leeb-Lundberg
authors.forename: Merle S
authors.initials: MS
authors.lastname: Olson
authors.forename: Christine
authors.initials: C
authors.lastname: Saunders
mesh_terms: D019390:Arrestins / Q000378:metabolism; D015153:Blotting, Western; D002122:Calcium Chloride / Q000494:pharmacology; D002455:Cell Division; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D015151:Immunoblotting; D020935:MAP Kinase Signaling System; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D000071557:beta-Arrestins; D003208:Concanavalin A; D037342:Pertussis Toxin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002122:Calcium Chloride