Mechanism of the desensitization of MC4R

MC4R的脱敏机制

• 批准号: 8396001
• 负责人: Brent Matthew Molden
• 金额: $ 3.44万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8396001
• 关键词: Accounting; Adenylate Cyclase; Adult; Affinity Chromatography; Agonist; Alanine; Antibodies; Back; Binding; Biological Assay; Cardiovascular Diseases; Cell membrane; Cell surface; Cells; Co-Immunoprecipitations; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Desire for food; Drug Delivery Systems; Drug usage; Endocytosis; Energy Metabolism; Exposure to; G protein coupled receptor kinase; G-Protein-Coupled Receptors; Genes; Health; Health Benefit; Health Expenditures; Homeostasis; Human; Hypothalamic structure; Incubated; Knockout Mice; Lead; Link; MAPK3 gene; Mass Spectrum Analysis; Measures; Melanocortin 4 Receptor; Melanocyte stimulating hormone; Membrane Potentials; Modeling; Morbidity - disease rate; Mutate; Mutation; Neurons; Obesity; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Prevention; Production; Protein Dephosphorylation; Proteins; Public Health; Radioactive; Receptor Activation; Recycling; Role; Sampling; Satiation; Signal Pathway; Signal Transduction; Small Interfering RNA; Sorting - Cell Movement; Surface; United States; Western Blotting; alpha-Melanocyte stimulating hormone; base; controlled release; desensitization; diabetes risk; early onset; effective therapy; human GPR4 protein; increased appetite; inorganic phosphate; mortality; mutant; novel; obesity treatment; paraventricular nucleus; receptor; receptor binding; receptor expression; receptor recycling; response; therapeutic target; trafficking

DESCRIPTION (provided by applicant): The Melanocortin Receptor 4 (MC4R) is a G-protein coupled receptor (GPCR) which is central to the control of appetite. Upstream signaling pathways converge to control release of the agonist of MC4R, alpha-melanocyte stimulating hormone (MSH), which transduces the signal of satiety upon binding to the receptor. Like other GPCRs, MC4R is desensitized upon agonist stimulation, leading to decreased response to a subsequent signal of equal magnitude. Our lab has shown that MC4R is not desensitized according to the canonical desensitization mechanism of most GPCRs. Classically, GPCRs are phosphorylated and rapidly endocytosed upon agonist stimulation. As we have shown, MC4R is internalized constitutively, at the same rate in the presence and absence of the agonist. Upon agonist stimulation, less receptor is recycled back to the plasma membrane, likely accounting for decreased expression of the receptor at the cell surface. I have shown that the decrease in surface receptor upon incubation with MSH leads to a corresponding decrease in cAMP production in the wild type MC4R. Conversely, a mutated MC4R with presumed phosphorylation sites mutated to alanine has similar cell surface expression, recycles back to the plasma membrane at the same rate, and, as I have shown, produces equivalent levels of cAMP regardless of treatment with the agonist. This result suggests that receptor phosphorylation is necessary to sequester the receptor in intracellular compartments in the presence of the agonist. Because desensitization of MC4R would lead to increased appetite, inhibiting factors which function in this pathway is a potential target for obesity drugs. In this study, we will idenify factors involved in sorting phosphorylated MC4R away from recycling pathways and the kinase(s) that phosphorylate MC4R. In Aim1 we will perform an affinity purification of a stably transfected HA-MC4R-GFP and determine interacting factors via mass spectrometry. Co-purified proteins will be investigated for their possible effect on MC4R traffic and desensitizatio. In Aim 2, we will silence or pharmacologically inhibit G- Protein Coupled Receptor Kinases (GRKs) and kinases that have been previously described to be activated upon MC4R activation, and measure the effect on the phosphorylation state, plasma membrane expression and activity of MC4R. By identifying and factors involved in sequestering it in intracellular compartments, and factors that phosphorylate MC4R we will create drug targets for much needed obesity therapy. PUBLIC HEALTH RELEVANCE: Obesity is an increasingly important health problem in the United States and worldwide. Although effective treatments for the prevention and reversal of obesity would have an enormous public health benefit, currently available therapies are largely ineffective or unsafe. Here, we propose to study the desensitization of the Melanocortin Receptor 4, a protein central to the control of appetite and energy expenditure, in an attempt to identify novel drug targets for the prevention and treatment of obesity.

描述（由申请人提供）：黑色素皮质素受体4（MC4R）是G蛋白偶联受体（GPCR），是食欲控制的核心。上游信号通路会收敛到控制MC4R激动剂，α-甲状腺细胞刺激激素（MSH）的激动剂的释放，该激素刺激激素（MSH）在与受体结合时会传递饱腹感的信号。像其他GPCR一样，MC4R在激动剂刺激下脱敏，从而导致对随后的相等幅度信号的响应减少。我们的实验室表明，根据大多数GPCR的规范脱敏机制，MC4R不会脱敏。从经典上讲，GPCR被磷酸化并在激动剂刺激下迅速内吞。正如我们已经表明的那样，在存在和不存在激动剂的情况下，MC4R以相同的速率进行组成式化。激动剂刺激后，较少的受体被回收回到质膜，这可能会使受体在细胞表面的表达降低。我已经表明，与MSH孵育后，表面受体的降低导致野生型MC4R的cAMP产生相应减少。相反，突变为突变为丙氨酸的磷酸化位点的突变MC4R具有相似的细胞表面表达，以相同的速率回收回到质膜，并且正如我所表明的那样，无论对激动剂的治疗方法如何，都会产生同等水平的cAMP。该结果表明，在激动剂存在下，在细胞内室中隔离受体是必要的。由于MC4R的脱敏会导致食欲增加，因此在该途径中起作用的抑制因素是肥胖药物的潜在靶标。在这项研究中，我们将鉴于从回收途径和磷酸化MC4R的激酶中分类磷酸化的MC4R的因素。在AIM1中，我们将对稳定转染的HA-MC4R-GFP进行亲和力纯化，并通过质谱确定相互作用的因子。将研究共纯化的蛋白质对MC4R流量和去脱义的可能影响。在AIM 2中，我们将在MC4R激活时被描述为被描述为被激活的G-蛋白偶联受体激酶（GRKS）和激酶的静音或药理抑制，并测量对磷酸化状态，质膜膜表达和MC4R活性的影响。通过识别在细胞内室中隔离IT的因素以及磷酸化MC4R的因素，我们将为急需的肥胖治疗创建药物靶标。 公共卫生相关性：肥胖是美国和全球范围内越来越重要的健康问题。尽管有效的预防肥胖症治疗方法将具有巨大的公共卫生益处，但目前可用的疗法在很大程度上是无效或不安全的。在这里，我们建议研究黑色素皮质受体4的脱敏，这是一种控制食欲和能量消耗的核心，以识别预防和治疗肥胖症的新药物靶标。