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首页> 外文期刊>Biochemistry >Transmembrane segment peptides can disrupt cholecystokinin receptor oligomerization without affecting receptor function
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Transmembrane segment peptides can disrupt cholecystokinin receptor oligomerization without affecting receptor function

机译：跨膜段肽可以破坏胆囊收缩素受体的寡聚而不影响受体功能

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摘要

Oligomerization of the G protein-coupled cholecystokinin (CCK) receptor has been demonstrated, but its molecular basis and functional importance are not clear. We now examine contributions of transmembrane (TM) segments to oligomerization of this receptor using a peptide competitive inhibition strategy. Oligomerization of CCK receptors tagged at the carboxyl terminus with Renilla luciferase or yellow fluorescent protein was quantified using bioluminescence resonance energy transfer (BRET). Synthetic peptides representing TM I, II, V, VI, and VII of the CCK receptor were utilized as competitors. Of these, only TM VI and VII peptides disrupted receptor BRET. Control studies established that the beta(2)-adrenergic receptor TM VI peptide that disrupts oligomerization of that receptor had no effect on CCK receptor BRET. Notably, disruption of CCK receptor oligomerization had no effect on agonist binding, biological activity, or receptor internalization. To gain insight into the face of TM VI contributing to oligomerization, we utilized analogous peptides with alanines in positions 315, 319, and 323 (interhelical face) or 317, 321, and 325 (external lipid-exposed face). The Ala(317,321,325) peptide eliminated the disruptive effect on CCK receptor BRET, whereas the other mutant peptide behaved like wild-type TM VI. This suggests that the lipid-exposed face of the CCK receptor TM VI most contributes to oligomerization and supports external contact dimerization of helical bundles, rather than domain-swapped dimerization. Fluorescent CCK receptor mutants with residues 317, 321, and 325 replaced with alanines were also prepared and failed to yield significant resonance transfer signals using either BRET or a morphological FRET assay, further supporting this interpretation.
机译：已证明G蛋白偶联的胆囊收缩素（CCK）受体的低聚作用，但其分子基础和功能重要性尚不清楚。现在，我们使用肽竞争性抑制策略研究跨膜（TM）段对该受体的寡聚作用。使用生物发光共振能量转移（BRET）定量标记在肾末端羧基标记的CCK受体与海肾荧光素酶或黄色荧光蛋白的寡聚。代表CCK受体的TM I，II，V，VI和VII的合成肽被用作竞争者。其中，只有TM VI和VII肽破坏受体BRET。对照研究确定，破坏该受体低聚的β（2）-肾上腺素受体TM VI肽对CCK受体BRET没有影响。值得注意的是，CCK受体低聚的破坏对激动剂结合，生物学活性或受体内在化没有影响。为了深入了解TM VI有助于寡聚化的表面，我们使用了在315、319和323（螺旋间表面）或317、321和325（外部脂质暴露的表面）位置具有丙氨酸的类似肽。 Ala（317,321,325）肽消除了对CCK受体BRET的破坏作用，而其他突变肽的行为类似于野生型TM VI。这表明CCK受体TM VI的脂质暴露面最有助于寡聚并支持螺旋束的外部接触二聚化，而不是结构域交换的二聚化。还制备了由丙氨酸取代的残基317、321和325的荧光CCK受体突变体，使用BRET或形态学FRET分析无法产生明显的共振转移信号，进一步支持了这一解释。

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