Miettinen H M, Mills J S, Gripentrog J M, Dratz E A, Granger B L, Jesaitis A J
Department of Microbiology, Montana State University, Bozeman 59717, USA.
J Immunol. 1997 Oct 15;159(8):4045-54.
We propose that the N-formyl-Met-Leu-Phe binding site in the human neutrophil formyl peptide receptor (FPR) lies in the predicted transmembrane region. We examined the expression, binding, and G protein coupling of 28 mutated forms of FPR in stably transfected Chinese hamster ovary cells. The amino acids we mutated are: 1) predicted to be oriented toward the interhelical space; 2) analogous to those required for ligand binding in various other G protein-coupled receptors; 3) divergent from lipoxin A4 receptor, a low affinity receptor for formylated peptides; and 4) either highly conserved or divergent in other G protein-coupled receptors. Some mutations resulted in intracellular retention, suggesting that the receptors were misfolded. Most mutated receptors that were transported to the plasmalemma bound f-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with affinities similar to the wild-type receptor (Kd = 6 nM). However, mutations L78A (helix II), D106N, L109A (helix III), T157A (helix IV), R201A, I204Y, and R205A (helix V), W254A and Y257A (helix VI), and F291A (helix VII) resulted in reduced affinities (Kd = 30-128 nM). Of these mutations, D106N, R201A, and R205A also appeared to affect G protein coupling, suggesting that these residues may also be involved in signal transduction and/or are essential for proper folding of the molecule. Some of the FPR residues that appeared to be involved in binding of formylated peptides were located at sites analogous to those identified in ligand binding to certain other G protein-coupled receptors. It is thus possible that several G protein-coupled receptors have a common placement of ligand-binding amino acids.
我们提出，人类中性粒细胞甲酰肽受体（FPR）中的N-甲酰基-蛋氨酸-亮氨酸-苯丙氨酸结合位点位于预测的跨膜区域。我们检测了28种FPR突变形式在稳定转染的中国仓鼠卵巢细胞中的表达、结合及G蛋白偶联情况。我们突变的氨基酸有：1）预测朝向螺旋间空间；2）类似于其他各种G蛋白偶联受体中配体结合所需的氨基酸；3）与脂氧素A4受体不同，脂氧素A4受体是一种对甲酰化肽低亲和力的受体；4）在其他G蛋白偶联受体中高度保守或不同。一些突变导致细胞内滞留，表明受体折叠错误。大多数转运到质膜的突变受体与f-Nle-Leu-Phe-Nle-Tyr-Lys-荧光素结合，其亲和力与野生型受体相似（Kd = 6 nM）。然而，L78A（螺旋II）、D106N、L109A（螺旋III）、T157A（螺旋IV）、R201A、I204Y和R205A（螺旋V）、W254A和Y257A（螺旋VI）以及F291A（螺旋VII）突变导致亲和力降低（Kd = 30 - 128 nM）。在这些突变中，D106N、R201A和R205A似乎也影响G蛋白偶联，表明这些残基可能也参与信号转导和/或对分子的正确折叠至关重要。一些似乎参与甲酰化肽结合的FPR残基位于与某些其他G蛋白偶联受体配体结合位点类似的位置。因此，几种G蛋白偶联受体可能具有共同的配体结合氨基酸位置。
