TY - JOUR
T1 - Activity of adenylyl cyclase type 6 is suppressed by direct binding of the cytoskeletal protein 4.1G
AU - Saito, Masaki
AU - Cui, Linran
AU - Hirano, Marina
AU - Li, Guanjie
AU - Yanagisawa, Teruyuki
AU - Sato, Takeya
AU - Sukegawa, Jun
N1 - Funding Information:
This work was supported by the Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (No. 15K20856 and No. 18K06125 to M.S., No. 24590311 to T.Y., No. 18K06885 to T.S., and No. 21200007, No. 21590267, No. 26460331 to J.S.), Takeda Science Foundation, Nishinomiya Basic Research Fund (to M.S.), and Shokei Gakuin University (to J.S.). Conflict of interest: The authors declare that they have no conflict of interest. https://doi.org/10.1124/mol.119.116426. s This article has supplemental material available at molpharm. aspetjournals.org.
Funding Information:
This work was supported by the Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (No. 15K20856 and No. 18K06125 to M.S., No. 24590311 to T.Y., No. 18K06885 to T.S., and No. 21200007, No. 21590267, No. 26460331 to J.S.), Takeda Science Foundation, Nishinomiya Basic Research Fund (to M.S.), and Shokei Gakuin University (to J.S.). Conflict of interest: The authors declare that they have no conflict of interest. We greatly appreciate Dr. Colin Taylar (University of Cambridge, UK), Dr. Shotaro Tanaka (Tokyo Women?s Medical University, Japan), and National Institute of Technology and Evaluation for providing pEGFP-C2-AC6-FL, pcDNA4-DsRed-TDM and pME18SF3-AC3-FL-WT plasmids, respectively. We would also like to acknowledge the Biomedical Research Core (Tohoku University Graduate School of Medicine) and Sayuri Saito for technical support.
Publisher Copyright:
Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.
PY - 2019/10
Y1 - 2019/10
N2 - The G protein-coupled receptor (GPCR) signaling pathways mediated by trimeric G proteins have been extensively elucidated, but their associated regulatory mechanisms remain unclear. Parathyroid hormone (PTH)/PTH-related protein receptor (PTHR) is a GPCR coupled with Gs and Gq. Gs activates adenylyl cyclases (ACs), which produces cAMP to regulate various cell fates. We previously showed that cell surface expression of PTHR was increased by its direct interaction with a subcortical cytoskeletal protein, 4.1G, whereas PTHR-mediated Gs/AC/cAMP signaling was suppressed by 4.1G through an unknown mechanism in human embryonic kidney (HEK)293 cells. In the present study, we found that AC type 6 (AC6), one of the major ACs activated downstream of PTHR, interacts with 4.1G in HEK293 cells, and the N-terminus of AC6 (AC6-N) directly and selectively binds to the 4.1/ezrin/radixin/moesin (FERM) domain of 4.1G (4.1G-FERM) in vitro. AC6-N was distributed at the plasma membrane, which was disturbed by knockdown of 4.1G. An AC6-N mutant, AC6-N-3A, in which three consecutive arginine residues are mutated to alanine residues, altered both binding to 4.1G-FERM and its plasma membrane distribution in vivo. Further, we overexpressed AC6-N to competitively inhibit the interaction of endogenous AC6 and 4.1G in cells. cAMP production induced by forskolin, an adenylyl cyclase activator, and PTH-(1-34) was enhanced by AC6-N expression and 4.1G-knockdown. In contrast, AC6-N-3A had no impact on forskolin- and PTH-(1-34)-induced cAMP productions. These data provide a novel regulatory mechanism that AC6 activity is suppressed by the direct binding of 4.1G to AC6-N, resulting in attenuation of PTHR-mediated Gs/AC6/cAMP signaling.
AB - The G protein-coupled receptor (GPCR) signaling pathways mediated by trimeric G proteins have been extensively elucidated, but their associated regulatory mechanisms remain unclear. Parathyroid hormone (PTH)/PTH-related protein receptor (PTHR) is a GPCR coupled with Gs and Gq. Gs activates adenylyl cyclases (ACs), which produces cAMP to regulate various cell fates. We previously showed that cell surface expression of PTHR was increased by its direct interaction with a subcortical cytoskeletal protein, 4.1G, whereas PTHR-mediated Gs/AC/cAMP signaling was suppressed by 4.1G through an unknown mechanism in human embryonic kidney (HEK)293 cells. In the present study, we found that AC type 6 (AC6), one of the major ACs activated downstream of PTHR, interacts with 4.1G in HEK293 cells, and the N-terminus of AC6 (AC6-N) directly and selectively binds to the 4.1/ezrin/radixin/moesin (FERM) domain of 4.1G (4.1G-FERM) in vitro. AC6-N was distributed at the plasma membrane, which was disturbed by knockdown of 4.1G. An AC6-N mutant, AC6-N-3A, in which three consecutive arginine residues are mutated to alanine residues, altered both binding to 4.1G-FERM and its plasma membrane distribution in vivo. Further, we overexpressed AC6-N to competitively inhibit the interaction of endogenous AC6 and 4.1G in cells. cAMP production induced by forskolin, an adenylyl cyclase activator, and PTH-(1-34) was enhanced by AC6-N expression and 4.1G-knockdown. In contrast, AC6-N-3A had no impact on forskolin- and PTH-(1-34)-induced cAMP productions. These data provide a novel regulatory mechanism that AC6 activity is suppressed by the direct binding of 4.1G to AC6-N, resulting in attenuation of PTHR-mediated Gs/AC6/cAMP signaling.
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U2 - 10.1124/mol.119.116426
DO - 10.1124/mol.119.116426
M3 - Article
C2 - 31383768
AN - SCOPUS:85071782523
SN - 0026-895X
VL - 96
SP - 441
EP - 451
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 4
ER -