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.
Highlights
¶ Current address: Division of Molecular Neurobiology, Wallenberg Neuroscience Center, Lund University, BMC, A12, SE-22184 Lund, Sweden
There was no significant desensitization to calcium-sensing receptor (CaR) stimulation of ERK1/2 phosphorylation when 1 M NPS R-467 in the presence of 0.5 mM CaCl2 was used as agonists, even as late as 240 min after the addition of stimulus (Fig. 2B)
In this study we demonstrate that NPS R-467-mediated ERK1/2 activation via the CaR does not appear to undergo desensitization compared with the activity induced by a high concentration of Ca2ϩ alone
Summary
¶ Current address: Division of Molecular Neurobiology, Wallenberg Neuroscience Center, Lund University, BMC, A12, SE-22184 Lund, Sweden. The CaR has been reported to activate Kϩ channels [15] and produce a pertussis toxin-sensitive (Gi) inhibition of agonist-stimulated cAMP accumulation in some cells [16]. These findings suggest that the CaR can couple to more than one population of G proteins. The mitogen-activated protein kinase, MAP kinase, pathways have been described to regulate a number of cell processes such as cell growth, differentiation, apoptosis, oncogenetic transformation, and enzyme activity [17] These include the extracellular signal-regulated kinases (ERKs) that are stimulated by agonist binding to GPCR and tyrosine kinase growth factor receptors as well as by mechanical stress in many cell types. It has been determined that the dose-response curve for the ability of calcium to activate down-stream signaling cascades (intracellular calcium mobilization, ERK1/2 activation) in cells expressing the CaR is shifted to the left at least 10-fold in the presence of sis toxin; HSGB, high salt glucose buffer; PBS, phosphate-buffered saline; EGF, epidermal growth factor
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