大麻素受体相互作用蛋白1a调节CB1受体信号传导与调控。
Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.
作者信息
Smith Tricia H, Blume Lawrence C, Straiker Alex, Cox Jordan O, David Bethany G, McVoy Julie R Secor, Sayers Katherine W, Poklis Justin L, Abdullah Rehab A, Egertová Michaela, Chen Ching-Kang, Mackie Ken, Elphick Maurice R, Howlett Allyn C, Selley Dana E
机构信息
Department of Pharmacology and Toxicology and Institute for Drug and Alcohol Studies (T.H.S., J.O.C., B.G.D., J.R.S.M., J.L.P., R.A.A., D.E.S.), Department of Anatomy and Neurobiology (K.W.S.), Department of Biochemistry and Molecular Biology (C.-K.C.), Virginia Commonwealth University School of Medicine, Richmond, Virginia; Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina (L.C.B., A.C.H.); The Gill Center for Biomolecular Science and the Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana (A.S., K.M.); and School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom (M.E., M.R.E.).
Department of Pharmacology and Toxicology and Institute for Drug and Alcohol Studies (T.H.S., J.O.C., B.G.D., J.R.S.M., J.L.P., R.A.A., D.E.S.), Department of Anatomy and Neurobiology (K.W.S.), Department of Biochemistry and Molecular Biology (C.-K.C.), Virginia Commonwealth University School of Medicine, Richmond, Virginia; Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina (L.C.B., A.C.H.); The Gill Center for Biomolecular Science and the Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana (A.S., K.M.); and School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom (M.E., M.R.E.)
出版信息
Mol Pharmacol. 2015 Apr;87(4):747-65. doi: 10.1124/mol.114.096495. Epub 2015 Feb 5.
Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.
大麻素CB1受体（CB1Rs）介导内源性大麻素在中枢神经系统（CNS）中的突触前效应以及外源性大麻素的大多数行为效应。大麻素受体相互作用蛋白1a（CRIP1a）与CB1R的C末端结合，并可减弱CB1R介导的对Ca(2+)通道活性的组成性抑制。我们现在证明了CRIP1a在CNS神经元成分中的细胞共定位，并表明CRIP1a抑制组成性和激动剂刺激的CB1R介导的鸟嘌呤核苷酸结合调节蛋白（G蛋白）活性。在稳定表达CB1Rs的人胚肾（HEK）-293细胞（CB1-HEK）中，或在内源性表达CB1Rs的N18TG2细胞中稳定过表达CRIP1a，均降低了两种细胞系中CB1R介导的G蛋白激活（通过激动剂刺激的[(35)S]GTPγS（鸟苷-5'-[O-硫代]-三磷酸）结合来测量），并减弱了利莫那班在CB1-HEK细胞中的反向激动作用。相反，在N18TG2细胞中，小干扰RNA介导敲低CRIP1a增强了CB1R介导的G蛋白激活。这些效应并非归因于CB1R表达或内源性大麻素水平的差异，因为在CRIP1a表达不同的细胞系之间，CB1R水平没有差异，并且过表达CRIP1a后，内源性大麻素水平在CB1-HEK细胞中无法检测到，在N-18TG2细胞中未发生变化。在CB1-HEK细胞中，用大麻素激动剂预处理4小时可下调CB1Rs并使激动剂刺激的[(35)S]GTPγS结合脱敏。CRIP1a过表达减弱了CB1R下调，但未改变CB1R脱敏。最后，在培养的自突触海马神经元中，CRIP1a过表达减弱了去极化诱导的兴奋抑制以及外源性应用大麻素而非腺苷A1激动剂诱导的兴奋性突触活动抑制。这些结果证实CRIP1a抑制组成性CB1R活性，并表明CRIP1a也可抑制激动剂刺激的CB1R信号传导和CB1Rs的下调。因此；CRIP1a似乎是CB1R功能的广泛负调节因子。
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