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Proc Natl Acad Sci U S A. 2019 Aug 6;116(32):15895-15900. doi: 10.1073/pnas.1818547116. Epub 2019 Jul 23.

Perturbation of the interactions of calmodulin with GRK5 using a natural product chemical probe.

Author information

1
Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109.
2
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109.
3
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109.
4
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
5
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907.
6
Department of Pharmacology, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140.
7
Center for Translational Medicine, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140.
8
Department of Chemistry, College of Natural Sciences, Colorado State University, Fort Collins, CO 80523.
9
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109.
10
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109.
11
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907; jtesmer@purdue.edu.

Abstract

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca2+·CaM-GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca2+·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca2+·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca2+·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca2+·CaM-dependent hypertrophy.

KEYWORDS:

G protein-coupled receptor kinase 5; calmodulin; hypertrophy; malbrancheamide

PMID:
31337679
PMCID:
PMC6689901
[Available on 2020-01-23]
DOI:
10.1073/pnas.1818547116

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