Di Paolo E, Vilardaga J P, Petry H, Moguilevsky N, Bollen A, Robberecht P, Waelbroeck M
Department of Biochemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Belgium.
Peptides. 1999;20(10):1187-93. doi: 10.1016/s0196-9781(99)00122-9.
The secretin receptor is a member of a large family of G-protein-coupled receptors that recognize polypeptide hormone and/or neuropeptides. Charged, conserved residues might play a key role in their function, either by interacting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor family, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D severely reduced both the maximal response to secretin and its potency. No functional secretin receptor could be detected after replacement of R83 by L. Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellular ends of TM III (K194) and IV (R255) were important for the secretin receptor function, as K194 mutation to A or Q and R255 mutation to Q or D decreased the secretin's affinity 15- to 1000-fold, respectively. Six extracellular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for functional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to stimulate normally the adenylate cyclase in response to secretin, and functional D174A receptors could not be found. Mutation of R255, E259 (second extracellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its efficacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (TM I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor functional properties.
促胰液素受体是识别多肽激素和/或神经肽的G蛋白偶联受体大家族的成员。带电荷的保守残基可能通过与配体相互作用或稳定受体结构在其功能中发挥关键作用。在整个促胰液素受体家族中保守的四个带电荷氨基酸中，D49和R83（在N端结构域）可能对促胰液素受体结构很重要：用H或R取代D49以及用D取代R83会严重降低对促胰液素的最大反应及其效力。用L取代R83后未检测到有功能的促胰液素受体。将D49突变为E、A或N没有影响或使促胰液素的效力降低了5倍。在跨膜区III（K194）和IV（R255）的细胞外末端发现的高度保守的正电荷对促胰液素受体功能很重要，因为将K194突变为A或Q以及将R255突变为Q或D分别使促胰液素的亲和力降低了15至1000倍。六个细胞外带电荷残基在密切相关的受体中保守，但在整个家族中并非如此。K121（跨膜区I）和R277（跨膜区V）对功能性促胰液素受体表达并不重要。D174（跨膜区II）对于稳定活性受体结构是必需的：D174N突变体受体不能正常响应促胰液素刺激腺苷酸环化酶，并且未发现有功能的D174A受体。将R255、E259（第二个细胞外环）和E351（第三个细胞外环）突变为不带电荷的残基仅使促胰液素效力降低了10至100倍，而不改变其功效：这些残基要么稳定活性受体构象，要么与促胰液素形成氢键而非离子键。将K121（跨膜区I）突变为Q或L以及将R277（跨膜区V）突变为E或Q不影响受体功能特性。
