Mechanism of the desensitization of MC4R

MC4R的脱敏机制

基本信息

批准号：
8499962
负责人：
Brent Matthew Molden
金额：
$ 4.15万
依托单位：
UNIV OF ARKANSAS FOR MED SCIS
依托单位国家：
美国
项目类别：
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/8499962
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 Targeting 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

项目摘要

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.

描述（由申请人提供）：黑皮质素受体 4 (MC4R) 是一种 G 蛋白偶联受体 (GPCR)，对于控制食欲至关重要。上游信号通路汇聚以控制 MC4R 激动剂（α-黑素细胞刺激激素 (MSH)）的释放，该激动剂在与受体结合后转导饱腹感信号。与其他 GPCR 一样，MC4R 在激动剂刺激下会脱敏，导致对后续同等强度信号的反应减弱。我们的实验室已经表明，根据大多数 GPCR 的典型脱敏机制，MC4R 并未脱敏。传统上，GPCR 在激动剂刺激下被磷酸化并快速内吞。正如我们所表明的，MC4R 在存在和不存在激动剂的情况下以相同的速率被组成性内化。在激动剂刺激后，较少的受体被再循环回质膜，这可能是细胞表面受体表达减少的原因。我已经证明，与 MSH 孵育后表面受体的减少会导致野生型 MC4R 中 cAMP 产量的相应减少。相反，假定磷酸化位点突变为丙氨酸的突变 MC4R 具有相似的细胞表面表达，以相同的速率循环回质膜，并且如我所示，无论是否使用激动剂进行处理，都会产生相同水平的 cAMP。该结果表明，在激动剂存在的情况下，受体磷酸化对于将受体隔离在细胞内区室中是必要的。由于 MC4R 脱敏会导致食欲增加，因此在该途径中发挥作用的抑制因子是肥胖药物的潜在目标。在本研究中，我们将鉴定参与将磷酸化 MC4R 从回收途径中分选出来的因素以及磷酸化 MC4R 的激酶。在 Aim1 中，我们将对稳定转染的 HA-MC4R-GFP 进行亲和纯化，并通过质谱法确定相互作用因子。将研究共纯化的蛋白质对 MC4R 流量和脱敏的可能影响。在目标 2 中，我们将沉默或药理学抑制先前描述的在 MC4R 激活时被激活的 G 蛋白偶联受体激酶 (GRK) 和激酶，并测量对 MC4R 磷酸化状态、质膜表达和活性的影响。通过识别将其隔离在细胞内区室中所涉及的因素以及磷酸化 MC4R 的因素，我们将为急需的肥胖治疗创建药物靶点。