Dalman H M, Neubig R R
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
J Biol Chem. 1991 Jun 15;266(17):11025-9.
Peptides derived from various regions of the alpha 2A-adrenergic receptor (alpha 2A-AR) were used to study receptor-G protein interactions. Binding of the partial agonist [125I]-p-iodoclonidine and the full agonist [3H]bromoxidine (UK14,304) to membrane preparations from human platelet was potently reduced by peptides (12-14 amino acids) from the second cytoplasmic loop (A) and the C-terminal side of the third cytoplasmic loop (Q). Binding of the antagonist [3H]yohimbine was significantly less affected. Five other peptides had no significant effects on ligand binding at concentrations less than 100 microM. The IC50 values for peptides A and Q were 7 and 27 microM for [125I]-p-iodoclonidine binding at the platelet alpha 2A receptor, 15 and 71 microM for the neuroblastoma-glioma (NG108-15) alpha 2B receptor, and greater than 300 microM for yohimbine binding at both alpha 2A and alpha 2B receptors. Competition studies demonstrate that at concentrations of 100 microM, peptides A and Q reduce the affinity of bromoxidine for the platelet alpha 2A-AR and this effect was abolished in the presence of guanine nucleotide. Alpha 2A-AR-stimulated GTPase activity in platelet membranes was inhibited by peptide Q with an IC50 of 16 microM but A was inactive. These data suggest that both the second cytoplasmic loop and the C-terminal part of the third cytoplasmic loop of the alpha 2A-AR are important in the interaction between the alpha 2-AR and Gi protein. Peptide Q appears to destabilize the high affinity state of the alpha 2-AR by binding directly to Gi thus preventing it from coupling to the receptor under both binding and GTPase assay conditions. The peptide from the second cytoplasmic loop (A) also reduces high affinity agonist binding in a G protein-dependent manner but its interaction with receptor and G protein is distinct in that it does not prevent activation of the G protein. These results provide new information about regions of the alpha 2-adrenergic receptor involved in G protein coupling and high affinity agonist binding.
来自α2A - 肾上腺素能受体（α2A - AR）不同区域的肽段被用于研究受体 - G蛋白相互作用。部分激动剂[125I]-对碘可乐定和完全激动剂[3H]溴辛定（UK14,304）与人血小板膜制剂的结合被来自第二胞质环（A）和第三胞质环C端侧（Q）的肽段（12 - 14个氨基酸）显著降低。拮抗剂[3H]育亨宾的结合受影响较小。其他五个肽段在浓度低于100μM时对配体结合无显著影响。肽段A和Q对血小板α2A受体上[125I]-对碘可乐定结合的IC50值分别为7μM和27μM，对神经母细胞瘤 - 胶质瘤（NG108 - 15）α2B受体上的IC50值分别为15μM和71μM，对α2A和α2B受体上育亨宾结合的IC50值均大于300μM。竞争研究表明，在100μM浓度下，肽段A和Q降低了溴辛定对血小板α2A - AR的亲和力，且这种作用在鸟嘌呤核苷酸存在时被消除。肽段Q抑制血小板膜中α2A - AR刺激的GTP酶活性，IC50为16μM，而肽段A无活性。这些数据表明，α2A - AR的第二胞质环和第三胞质环的C端部分在α2 - AR与Gi蛋白的相互作用中都很重要。肽段Q似乎通过直接与Gi结合来破坏α2 - AR的高亲和力状态，从而在结合和GTP酶测定条件下阻止其与受体偶联。来自第二胞质环的肽段（A）也以G蛋白依赖的方式降低高亲和力激动剂结合，但其与受体和G蛋白的相互作用不同，因为它不会阻止G蛋白的激活。这些结果提供了关于参与G蛋白偶联和高亲和力激动剂结合的α2 - 肾上腺素能受体区域的新信息。
