Beinborn M, Lee Y M, McBride E W, Quinn S M, Kopin A S
Division of Gastroenterology, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111.
Nature. 1993 Mar 25;362(6418):348-50. doi: 10.1038/362348a0.
The brain cholecystokinin-B/gastrin receptor (CCK-B/gastrin) has been implicated in mediating anxiety, panic attacks, satiety, and the perception of pain. The canine and human CCK-B/gastrin receptors share 90% amino-acid identity and have similar agonist affinities. These receptors can be selectively blocked by the non-peptide benzodiazepine-based antagonists L365260 (ref. 8) and L364718 (ref. 9); however, the binding of these antagonists to the human and canine receptors differs by up to 20-fold, resulting in a reversal of affinity rank order. Here we report the identification of a single amino acid in the sixth transmembrane domain of the CCK-B/gastrin receptor that corresponds to valine 319 in the human homologue and which is critical in determining the binding affinity for these non-peptide antagonists. We show that it is the variability in the aliphatic side chain of the amino acid in position 319 that confers antagonist specificity. Substitution of valine 319 with a leucine residue decreases the affinity for L365260 20-fold while concomitantly increasing the affinity for L364718. An isoleucine in the same position of the human receptor selectively increases affinity for L364718. Interspecies differences in the aliphatic amino acid occupying this single position selectively affect antagonist affinities without altering the agonist binding profile. We therefore conclude that the residues underlying non-peptide antagonist affinity must differ from those that confer agonist specificity. To our knowledge, these findings are the first example in which a critical antagonist binding determinant for a seven-transmembrane-domain peptide hormone receptor has been identified.
脑胆囊收缩素 -B/胃泌素受体（CCK -B/胃泌素）与焦虑、惊恐发作、饱腹感及疼痛感知的调节有关。犬类和人类的CCK -B/胃泌素受体有90%的氨基酸同源性，且具有相似的激动剂亲和力。这些受体可被非肽类苯二氮䓬类拮抗剂L365260（参考文献8）和L364718（参考文献9）选择性阻断；然而，这些拮抗剂与人及犬类受体的结合差异高达20倍，导致亲和力排序颠倒。在此，我们报告在CCK -B/胃泌素受体的第六跨膜结构域中鉴定出一个单一氨基酸，它与人同源物中的缬氨酸319相对应，且对决定这些非肽类拮抗剂的结合亲和力至关重要。我们发现，正是319位氨基酸脂肪族侧链的变异性赋予了拮抗剂特异性。将缬氨酸319替换为亮氨酸残基会使对L365260的亲和力降低20倍，同时使对L364718的亲和力增加。人类受体同一位置的异亮氨酸选择性地增加了对L364718的亲和力。占据这一单一位置的脂肪族氨基酸的种间差异选择性地影响拮抗剂亲和力，而不改变激动剂结合模式。因此，我们得出结论，非肽类拮抗剂亲和力的关键残基必定不同于赋予激动剂特异性的残基。据我们所知，这些发现是首次鉴定出七跨膜结构域肽激素受体关键拮抗剂结合决定因素的实例。
