Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC105
An AC8/Orai1 signalling complex underlies dynamic Ca2+-regulated cAMP signalling
D. Willoughby1, M. L. Halls1, K. L. Everett1, J. Pacheco2, L. Vaca2, P. Skroblin3, E. Klussmann4, D. M. Cooper1
1. Pharmacology, University of Cambridge, Cambridge, United Kingdom. 2. Departamento de Biolog
Ca2+ entry attributed to the activation of store-operated Ca2+ (SOC) channels within the plasma membrane, plays a critical role in numerous cellular functions. Orai1, a 33 kDa protein, is the pore-forming subunit of SOC channels (1,2). An important consequence of SOC channel mediated entry (SOCE) is the regulation of Ca2+-sensitive adenylyl cyclases (ACs). These ACs are responsive to sub-µM Ca2+ concentrations and are uniquely selective for SOCE over other modes of Ca2+ rise (3). Recent data from our laboratory using AC-targeted Ca2+ sensors has provided evidence of an intimate association between the Ca2+-stimulated AC8 and SOC channels in discrete ‘Ca2+ microdomains’ (4). The aim of our present study was to examine if there is a direct interaction between AC8 and Orai1 that underpins the functional dependence of AC8 on SOCE. All experiments were performed in cultured HEK293 cells. Using a multi-disciplinary approach of GST pull-downs, coimmunoprecipitation, FRET and peptide array analysis we demonstrate a direct binding between the N-termini of AC8 and Orai1. Specific sites of interactions were localized to a region within the first 106 residues of AC8 and an arginine-rich N-terminal region of Orai1 (residues 26-30). Live-cell imaging with AC8-targeted biosensors for Ca2+ (GCaMP2-AC8 (4)) or cAMP (Epac2-camps-AC8 (5)) was performed using a wide-field EM-CCD camera-based imaging system. Measurements obtained using GCaMP2-AC8 illustrated a rapid detection of SOCE with a time to peak of 16.4 ± 2.0 s (n=80 cells). In contrast, GCaMP2-AC8 detected little Ca2+ increase during 300µM CCh-evoked ER Ca2+ mobilization in Ca2+-free conditions. When GCaMP2 was tethered to an N-terminally truncated form of AC8 (GCaMP2-8M1, n=120 cells) the time to peak SOCE increased to 43.3 ± 1.8 s (p<0.0001, compared to GCaMP2-AC8). Ca2+ changes seen within the AC8-microdomain during SOCE were paralleled by enhanced cAMP production, detected by an AC8-targeted FRET-based cAMP biosensor, Epac2-camps-AC8. Knockdown of Orai1 expression attenuated the SOCE detected by GCaMP2-AC8 by 76 ± 4% (p<0.0001 compared to control) and AC8-mediated cAMP production by 61 ± 5% (p<0.001 compared to control). SOCE measured globally was reduced by > 90%, suggesting that any remaining Orai1 protein preferentially associates with AC8. We conclude that the longstanding functional dependence of AC8 on SOCE originates from a direct protein-protein interaction between AC8 and Orai1. Such intimacy ensures dynamic, coordinated changes in levels of Ca2+ and cAMP in cells expressing Ca2+-regulated ACs. This study provides the first demonstration that Orai1 can function as an integral component of larger signalling complexes and has major implications for how Ca2+-dependent signalling events may be organized.
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