Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor.
Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor.
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DOI：
10.1021/bi901326k
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发表时间：
2009-12-15
期刊：
影响因子：
2.9
通讯作者：
Miller, Laurence J.
中科院分区：
文献类型：
--
作者：
Harikumar, Kaleeckal G.;Simms, John;Christopoulos, George;Sexton, Patrick M.;Miller, Laurence J.
这三种受体活性修饰蛋白（RAMPs）已被认为对B - G蛋白偶联受体亚群的运输和功能起重要作用，尽管其结构基础尚未得到很好的确定。在目前的工作中，我们使用形态荧光技术、生物发光共振能量转移和双分子荧光互补来证明分泌素受体与RAMP3特异性结合，而不是与RAMP1或RAMP2特异性结合。我们使用截断构建、肽竞争实验和嵌合分泌素- glp1受体构建来确定这种关联是结构特异性的，依赖于RAMP的膜内区域和该受体的TM6和TM7。在存在或不存在外源性RAMP转染的情况下，在携带受体的COS或CHO-K1细胞中，分泌素刺激的cAMP、细胞内钙、ERK1/2磷酸化或受体内化均未观察到变化，尽管分泌素受体在这些细胞中以与RAMP无关的方式正常运输到细胞表面，导致细胞表面出现游离受体和RAMP相关受体。RAMP3与该受体的关联被证明能够挽救通常被困在生物合成机制中的细胞内的受体突变体（G241C）。同样，分泌素受体表达对肾上腺髓质素活性具有功能性影响，分泌素受体表达的增加与RAMP3与降钙素受体样受体相竞争，从而产生功能性肾上腺髓质素受体。这些数据为RAMP3与家族B G蛋白偶联受体相互作用的结构基础提供了重要的新见解，可能为药物作用提供高选择性靶点。这可能代表了该受体家族的其他成员与RAMP蛋白之间的类似相互作用。
The three receptor activity-modifying proteins (RAMPs) have been recognized as being important for the trafficking and function of a subset of family B G protein-coupled receptors, although the structural basis for this has not been well established. In the current work, we use morphological fluorescence techniques, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation to demonstrate that the secretin receptor associates specifically with RAMP3, but not with RAMP1 or RAMP2. We use truncation constructs, peptide competition experiments, and chimeric secretin-GLP1 receptor constructs to establish that this association is structurally-specific, dependent on the intramembranous region of the RAMP and TM6 and TM7 of this receptor. There were no observed changes in secretin-stimulated cAMP, intracellular calcium, ERK1/2 phosphorylation, or receptor internalization in receptor-bearing COS or CHO-K1 cells in the presence or absence of exogenous RAMP transfection, although the secretin receptor trafficks normally to the cell surface in these cells in a RAMP-independent manner, resulting in both free and RAMP-associated receptor on the cell surface. RAMP3 association with this receptor was shown to be capable of rescuing a receptor mutant (G241C) that is normally trapped intracellularly in the biosynthetic machinery. Similarly, secretin receptor expression had functional effects on adrenomedullin activity, with increasing secretin receptor expression competing for RAMP3 association with the calcitonin receptor-like receptor to yield a functional adrenomedullin receptor. These data provide important new insights into the structural basis for RAMP3 interaction with a family B G protein-coupled receptor, potentially providing a highly selective target for drug action. This may be representative of similar interactions between other members of this receptor family and RAMP proteins.
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DOI：
10.1080/10606820213683
发表时间：
2002-01-01
期刊：
RECEPTORS & CHANNELS
影响因子：
--
作者：
Born, W;Fischer, JA;Muff, R
通讯作者：
Muff, R
影响因子：
4.8
作者：
Bomberger, JM;Spielman, WS;Parameswaran, N
通讯作者：
Parameswaran, N
影响因子：
3.6
作者：
Dong, Maoqing;Lam, Polo C. -H.;Miller, Laurence J.
通讯作者：
Miller, Laurence J.
影响因子：
3.6
作者：
Dong, Maoqing;Lam, Polo C. -H.;Miller, Laurence J.
通讯作者：
Miller, Laurence J.
影响因子：
3.4
作者：
BenTal, N;Honig, B
通讯作者：
Honig, B