Identification of transmembrane regions critical for ligand binding to the human D3 dopamine receptor using various D3/D1 transmembrane chimeras
Identification of transmembrane regions critical for ligand binding to the human D3 dopamine receptor using various D3/D1 transmembrane chimeras
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DOI：
10.1124/mol.54.2.379
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发表时间：
1998-08-01
影响因子：
3.6
通讯作者：
Im, WB
中科院分区：
文献类型：
--
作者：
Alberts, GL;Pregenzer, JF;Im, WB
为了研究人D-3多巴胺受体的单个跨膜片段（TM）在其配体-受体相互作用中的作用，我们产生了嵌合受体，其中其TM被替换，一次一个，部分或全部，由相应的TM的同源人D-1受体。使用重组杆状病毒在Sf 9细胞中异源表达的嵌合体的配体结合特性表明，D-3选择性（超过D-1）配体的关键结合区域位于TMI、II、IV和VI的细胞外表面附近的狭窄区域（6至8个残基），而TMV似乎最低限度地参与配体选择性。对于TMIII和TMVII，关键区域似乎更深，对于TMIII，至少涉及细胞外表面附近的10个残基，对于TMVII，涉及整个TM区段。这是基于我们目前的观察结果，即在关键区域具有序列的嵌合体，尽管TM的其余部分是D-1来源的（除了TMVII），但显示出与野生型受体的结合特性无法区分。与D-1序列的嵌合体在该地区，另一方面，显示配体结合特性的变化很大，这取决于取代的TM：最显着的配体亲和力下降，观察与嵌合体的TMIII和VII，和中间的变化与TMIV和VI。TMV的替代对14种测试配体的亲和力没有明显的影响（除了一种）。嵌合体的TMI和II与D-1序列的关键区域没有表现出明显的特异性结合的几个放射性D-3-选择性配体，可能反映了他们的关键作用，在组装和折叠的受体。D-3受体的这些关键区域与D-2受体的那些区域高度同源，除了几个非保守取代的残基，其可以被利用来开发对D-3超过D-2多巴胺受体的选择性配体，反之亦然。
To investigate the roles of individual transmembrane segments (TM) of the human D-3 dopamine receptor in its ligand-receptor interactions, we generated chimeric receptors in which its TMs were replaced, one at a time, partially or entirely, by the corresponding TM of the homologous human D-1 receptor. Ligand binding properties of the chimeras, as expressed heterologously in Sf9 cells using recombinant baculoviruses, indicate that the critical binding regions for D-3-selective (over D-1) ligands reside at narrow regions (6 to 8 residues) near the extracellular surface for TMI, II, IV and VI, while TMV seems to be minimally involved in the ligand selectivity. For TMIII and TMVII, the critical regions seem to be deeper,involving at least the 10 residues near the extracellular surface for TMIII, and the entire TM segment for TMVII. This is based on our current observations that the chimeras with the, sequence in the critical regions, although the rest of the TM is of D-1 origin (except TMVII), showed the binding properties indistinguishable from those of the wild-type receptor. The chimeras with the D-1 sequence in the regions, on the other hand, showed ligand binding characteristics wildly variable depending on substituted TMs: Most marked decreases in ligand affinities were observed with the chimeras of TMIII and VII, and intermediate changes with those of TMIV and VI. Replacements of TMV produced no appreciable effects on the affinities of 14 test ligands (except for one). The chimeras of TMI and II with the D-1 sequence in the critical regions showed no appreciable specific binding for several radioactive D-3-selective ligands, possibly reflecting their critical roles in assembly and folding of the receptor. These critical regions of the D-3 receptor were highly homologous to those of the D-2 receptor, except for several nonconservatively substituted residues, which could be exploited to develop ligands selective for the D-3 over D-2 dopamine receptor or vice versa.