Beyermann M, Heinrich N, Fechner K, Furkert J, Zhang W, Kraetke O, Bienert M, Berger H
Leibniz-Institut für Molekulare Pharmakologie im FV Berlin e.V., Berlin, Germany.
Br J Pharmacol. 2007 Jul;151(6):851-9. doi: 10.1038/sj.bjp.0707293. Epub 2007 May 29.
Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands.
We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays.
Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did.
Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.
大多数药物作用于G蛋白偶联受体（GPCRs），这类受体通常能激活不同的信号转导事件。本研究旨在实现促肾上腺皮质激素释放因子1型受体（CRF(1)）配体的功能选择性。
我们用体积较大的氨基酸（苯甲酰苯丙氨酸、萘基丙氨酸）系统地取代urocortin（CRF(1)的天然肽类激动剂），并通过受体结合、[³⁵S]-GTPγS结合刺激和cAMP积累试验，测定这些类似物对人胚肾细胞中CRF(1)与Gs和Gi蛋白偶联作用的影响。
天然配体通过CRF(1)刺激Gs和Gi的激活，导致cAMP积累先被刺激后被抑制。urocortin第6至15位的单取代，根据取代基的位置和性质，产生了保留Gs活性但缺乏Gi活性的配体，这些配体仅刺激cAMP积累，并竞争性拮抗了蛙皮素对Gi的激活。相比之下与urocortin一样，该序列外有取代的类似物非选择性地激活Gs和Gi。
多肽激动剂urocortin中我们称为信号域的特定区域发生修饰后，产生的类似物可作为激动剂，同时也是CRF(1)激活不同G蛋白的拮抗剂。这一发现意味着受体与不同G蛋白偶联时活性构象存在显著差异。其他多肽激素受体配体中类似的信号信息结构编码，将为开发信号选择性候选药物带来一个通用概念。
