Purpose
This study was conducted to identify and characterize the structural requirements of a calmodulin-binding motif identified in the third intracellular (i3) loop of muscarinic acetylcholine receptors (M1–M5), a region important for G protein coupling.
Methods
GST fusion proteins and synthetic peptides derived from the hM1 i3 loop were tested for binding to CaM using a cross-linking gel shift assay and a dansyl-CaM fluorescence assay. Mutagenesis studies further characterized the structural requirements for the interaction and identified critical residues.
Results
28-Mer peptides from the C terminus of i3, representing the putative calmodulin domains of M1, M2, and M3, were found capable of interacting with CaM. In addition, smaller peptides defined a 5-amino-acid sequence essential for calmodulin binding. Studies performed with M1 peptides derived from GST fusion proteins, representing larger portions of the i3 C terminus, suggested the presence of a second adjacent CaM binding site. Mutagenesis studies identified two mutants that are unable to bind CaM: a point mutation, E360A, and a deletion mutant, Δ232–358.
Conclusion
Calmodulin can bind to an M1 region implicated in G protein coupling. This indicates an important role for CaM in the regulation of muscarinic signal transduction.
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Abbreviations
- CAm:
-
calmodulin
- GPCR:
-
G-protein coupled receptor
- hM1–3:
-
human muscarinic acetylcholine receptor, types 1–3
- mAchR:
-
muscarinic acetylcholine receptor
- mGluR:
-
metabotropic glutamate receptor
- MOR:
-
μ opioid receptor
- PKC:
-
protein kinase C
References
C. J. Koppen Particlevan B. Kaiser (2003) ArticleTitleRegulation of muscarinic acetylcholine receptor signaling Pharmacol. Ther. 98 197–220 Occurrence Handle10.1016/S0163-7258(03)00032-9 Occurrence Handle12725869
A. I. Levey C. A. Kitt W. F. Simonds D. L. Price M. R. Brann (1991) ArticleTitleIdentification and localization of muscarinic acetylcholine receptor proteins in brain with subtype-specific antibodies J. Neurosci. 11 3218–3226 Occurrence Handle1:CAS:528:DyaK3MXmsVymu7k%3D Occurrence Handle1941081
C. C. Felder A. C. Porter T. L. Skillman L. Zhang F. P. Bymaster N. M. Nathanson S. E. Hamilton J. Gomeza J. Wess D. L. McKinzie (2001) ArticleTitleElucidating the role of muscarinic receptors in psychosis Life Sci. 68 2605–2613 Occurrence Handle10.1016/S0024-3205(01)01059-1 Occurrence Handle1:CAS:528:DC%2BD3MXjs1WksL4%3D Occurrence Handle11392633
F. Dorje A. I. Levey M. R. Brann (1991) ArticleTitleImmunological detection of muscarinic receptor subtype proteins (m1–m5) in rabbit peripheral tissues Mol. Pharmacol. 40 459–462 Occurrence Handle1:STN:280:By2D3MjmslQ%3D Occurrence Handle1921982
S. E. Hamilton M. L. Schlador L. A. McKinnon R. S. Chmelar N. M. Nathanson (1998) ArticleTitleMolecular mechanisms for the regulation of the expression and function of muscarinic acetylcholine receptors J. Physiol. Paris 92 275–278 Occurrence Handle10.1016/S0928-4257(98)80032-6 Occurrence Handle1:CAS:528:DyaK1cXmsV2ms7w%3D Occurrence Handle9789822
S. E. Hamilton S. N. Hardouin S. G. Anagnostaras G. G. Murphy K. N. Richmond A. J. Silva E. O. Feigl N. M. Nathanson (2001) ArticleTitleAlteration of cardiovascular and neuronal function in m1 knockout mice Life Sci. 68 2489–2493 Occurrence Handle10.1016/S0024-3205(01)01043-8 Occurrence Handle1:CAS:528:DC%2BD3MXjs1Wksro%3D Occurrence Handle11392617
E. Bofill-Cardona O. Kudlacek Q. Yang H. Ahorn M. Friessmuth C. Nanoff (2000) ArticleTitleBinding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the G-protein activation switch J. Biol. Chem. 275 32672–32680 Occurrence Handle10.1074/jbc.M002780200 Occurrence Handle1:CAS:528:DC%2BD3cXnslOitLc%3D Occurrence Handle10926927
D. Wang W. Sadee J. M. Quillan (1999) ArticleTitleCalmodulin binding to G protein-coupling domain of opioid receptors J. Biol. Chem. 274 22081–22088 Occurrence Handle10.1074/jbc.274.31.22081 Occurrence Handle1:CAS:528:DyaK1MXltVymur0%3D Occurrence Handle10419536
R. Minakami N. Jinnai H. Sugiyama (1997) ArticleTitlePhosphorylation and calmodulin binding of the metabotropic glutamate receptor subtype 5 (mGluR5) are antagonistic in vitro J. Biol. Chem. 272 20291–20298 Occurrence Handle10.1074/jbc.272.32.20291 Occurrence Handle1:CAS:528:DyaK2sXltlGku7o%3D Occurrence Handle9242710
Y. Nakajima T. Yamamoto T. Nakayama S. Nakanishi (1999) ArticleTitleA relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7 J. Biol. Chem. 274 27573–27577 Occurrence Handle10.1074/jbc.274.39.27573 Occurrence Handle1:CAS:528:DyaK1MXmtlOks7k%3D Occurrence Handle10488094
J. H. Turner A. K. Gelasco J. R. Raymond (2004) ArticleTitleCalmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites J. Biol. Chem. 279 17027–17037 Occurrence Handle10.1074/jbc.M313919200 Occurrence Handle1:CAS:528:DC%2BD2cXjt1Ggtro%3D Occurrence Handle14752100
J. M. Quillan W. Sadee (1996) ArticleTitleStructure-based search for peptide ligands that cross-react with melanocortin receptor Pharm. Res. 13 1624–1630 Occurrence Handle10.1023/A:1016424203457 Occurrence Handle1:CAS:528:DyaK28XnsVKrtrc%3D Occurrence Handle8956325
Y. Zhang D. Wang W. Sadee (2005) ArticleTitleCalmodulin interaction with peptides from G-protein coupled receptors measured with S-tag labeling Biochem. Biophys. Res. Commun. 333 390–395 Occurrence Handle10.1016/j.bbrc.2005.05.129 Occurrence Handle1:CAS:528:DC%2BD2MXlsVChtb0%3D Occurrence Handle15950946
J. V. Frangioni B. G. Neel (1993) ArticleTitleSolubilization and purification of enzymatically active glutathione-S-transferase (pGEX) fusion proteins Anal. Biochem. 210 179–187 Occurrence Handle10.1006/abio.1993.1170 Occurrence Handle1:CAS:528:DyaK3sXkt1equ7Y%3D Occurrence Handle8489015
R. L. Kincaid M. Vaughn V. A. Tkachuk J. James C. Osborne (1982) ArticleTitleCa2+-dependent interaction of 5-dimethylaminoaphthalene-1-sulfonyl-calmodulin with cyclic nucleotide phosphodiesterase, calcineurin, and troponin I J. Biol. Chem. 257 10638–10643 Occurrence Handle1:CAS:528:DyaL38XlsFyltb4%3D Occurrence Handle6286665
C. G. Warr L. E. Kelly (1995) ArticleTitleIdentification and characterization of two distinct calmodulin-binding sites in the TrpI ion-channel protein of Drosophila melanogaster Biochem. J. 314 497–503
S. Maeda J. Lameh W. G. Mallet M. Phillip J. Ramachandran W. Sadee (1990) ArticleTitleInternalization of the hm1 muscarinic cholinergic receptor involves the third cytoplasmic loop FEBS J. 269 386–389 Occurrence Handle10.1016/0014-5793(90)81199-X Occurrence Handle1:CAS:528:DyaK3cXmtVGhsLY%3D
P. Hogger M. S. Shockley J. Lameh W. Sadee (1995) ArticleTitleActivating and inactivating mutations in N- and C-terminal i3 loop junctions of muscarinic acetylcholine hm1 receptors J. Biol. Chem. 270 7405–7410 Occurrence Handle10.1074/jbc.270.13.7405 Occurrence Handle1:CAS:528:DyaK2MXkvVOjs7k%3D Occurrence Handle7706286
M. A. Schumacher A. F. Rivard H. P. Bachinger J. Adelman (2001) ArticleTitleStructure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin Nature 410 1120–1124 Occurrence Handle10.1038/35074145 Occurrence Handle1:CAS:528:DC%2BD3MXjsVCrt70%3D Occurrence Handle11323678
F.-Y. Zeng J. Wess (1999) ArticleTitleIdentification and molecular characterization of m3 muscarinic receptor dimers J. Biol. Chem. 274 19487–19497 Occurrence Handle10.1074/jbc.274.27.19487 Occurrence Handle1:CAS:528:DyaK1MXksVWktbY%3D Occurrence Handle10383466
N. H. Lee N. S. M. Geoghagen E. Cheng R. T. Cline C. M. Fraser (1996) ArticleTitleAlanine scanning mutagenesis of conserved arginine/lysine–arginine/lysine-x-x-arginine/lysine G protein-activating motifs on m1 muscarinic acetylcholine receptors Mol. Pharmacol. 50 140–148 Occurrence Handle1:CAS:528:DyaK28XksVyls7g%3D Occurrence Handle8700106
M. Kataoka J. F. Head B. A. Seaton D. M. Engelman (1989) ArticleTitleMelittin binding causes a large calcium-dependent conformational change in calmodulin Proc. Natl. Acad. Sci. U.S.A. 86 6944–6948 Occurrence Handle1:CAS:528:DyaL1MXlvV2rsbg%3D Occurrence Handle2780551
T. Higashijima S. Uzu T. Nakajima E. M. Ross (1988) ArticleTitleMastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G-proteins) J. Biol. Chem. 263 6491–6494 Occurrence Handle1:CAS:528:DyaL1cXitFOmsbg%3D Occurrence Handle3129426
T. Higashijima J. Burnier E. M. Ross (1990) ArticleTitleRegulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines: mechanisms and structural determinants of activity J. Biol. Chem. 265 14176–14186 Occurrence Handle1:CAS:528:DyaK3cXmt1ygsL8%3D Occurrence Handle2117607
D. A. Malencik S. R. Anderson (1983) ArticleTitleHigh affinity binding of the mastoporans by calmodulin Biochem. Biophy. Res. Commun. 114 50–56 Occurrence Handle10.1016/0006-291X(83)91592-9 Occurrence Handle1:CAS:528:DyaL3sXks1KmtLg%3D
D. H. O'Day M. A. Myrc (2004) ArticleTitleCalmodulin-binding domains in Alzheimer's disease proteins: extending the calcium hypothesis Biochem. Biophy. Res. Commun. 320 1051–1054 Occurrence Handle10.1016/j.bbrc.2004.06.070
Z.-L. Lu J. W. Saldanha E. C. Hulme (2002) ArticleTitleSeven-transmembrane receptors: crystals clarify Trends Pharmacol. Sci. 23 140–146 Occurrence Handle10.1016/S0165-6147(00)01973-8 Occurrence Handle1:CAS:528:DC%2BD38XhsFCmsLk%3D Occurrence Handle11879682
Acknowledgment
This work was supported by NIH Research Grant DA04166, from the National Institute on Drug Abuse.
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Lucas, J.L., Wang, D. & Sadée, W. Calmodulin Binding to Peptides Derived from the i3 Loop of Muscarinic Receptors. Pharm Res 23, 647–653 (2006). https://doi.org/10.1007/s11095-006-9784-9
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DOI: https://doi.org/10.1007/s11095-006-9784-9