3 CYTOPLASMIC LOOPS OF RHODOPSIN INTERACT WITH TRANSDUCIN
3 CYTOPLASMIC LOOPS OF RHODOPSIN INTERACT WITH TRANSDUCIN
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DOI：
10.1073/pnas.86.18.6878
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发表时间：
1989-09-01
影响因子：
11.1
通讯作者：
HOFMANN, KP
中科院分区：
文献类型：
--
作者：
KONIG, B;ARENDT, A;HOFMANN, KP
视紫红质是一种古老的受体，通过与鸟嘌呤核苷酸结合蛋白（G蛋白）的相互作用传递信号。我们已经绘制了视紫红质与其G蛋白相互作用的位点，通过类比，这表明这类受体的其他成员可能与它们的G蛋白相互作用。视紫红质细胞质表面的三个区域与视杆细胞G蛋白转导素（Gt）相互作用。它们是（i）第二胞质环，其连接视紫红质螺旋III和IV，（ii）第三胞质环，其连接视紫红质螺旋V和VI，和（iii）由氨基酸310-321形成的推定的第四胞质环，因为羧基末端序列从螺旋VII出现并经由棕榈酰半胱氨酸322和323锚定到脂质双层。这些区域的视紫红质和GT的相互作用的证据来自于合成肽，包括这些区域的能力，以竞争与metarhodopsin II结合GT。光谱测定，测量的“额外的MII”引起的GT结合被用来测量GT的结合的程度在竞争肽的存在下。对应于第二，第三和第四细胞质环的三种肽有效地与后视紫红质II竞争，表现出在2 μ M范围内的Kd值; 11种另外的肽包括所有剩余的视紫红质表面区域，即使在200 μ M也未能竞争。作为有效竞争者的任何两种肽显示出协同效应，当混合时具有比单独测定时高15倍的有效性。一个数学模型被开发来描述这种行为。
Rhodopsin is a member of an ancient class of receptors that transduce signals through their interaction with guanine nucleotide-binding proteins (G proteins). We have mapped the sites of interaction of rhodopsin with its G protein, which by analogy suggests how other members of this class of receptors may interact with their G proteins. Three regions of rhodopsin's cytoplasmic surface interact with the rod cell G protein transducin (Gt). These are (i) the second cytoplasmic loop, which connects rhodopsin helices III and IV, (ii) the third cytoplasmic loop, which connects rhodopsin helices V and VI, and (iii) a putative fourth cytoplasmic loop formed by amino acids 310-321, as the carboxyl-terminal sequence emerges from helix VII and anchors to the lipid bilayer via palmitoylcysteines 322 and 323. Evidence for these regions of interaction of rhodopsin and Gt comes from the ability of synthetic peptides comprising these regions to compete with metarhodopsin II for binding to Gt. A spectroscopic assay that measures the "extra MII" caused by Gt binding was used to measure the extent of binding of Gt in the presence of competing peptides. The three peptides corresponding to the second, third, and fourth cytoplasmic loops competed effectively with metarhodopsin II, exhibiting Kd values in the 2 microM range; 11 additional peptides comprising all remaining surface regions of rhodopsin failed to compete even at 200 microM. Any two peptides that were effective competitors showed a synergistic effect, having 15 times higher effectiveness when mixed than when assayed separately. A mathematical model was developed to describe this behavior.