The second extracellular loop of the dopamine D₂receptor lines the binding-site crevice

Abstract

The binding site of the dopamine D2 receptor (D2R), like those of homologous rhodopsin-like G protein-coupled receptors (GPCRs) that bind small molecules, is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). The highresolution structure of bovine rhodopsin, however, revealed that the second extracellular loop (E2), which connects TM4 and TM5, folds down into the transmembrane domain and forms part of the ligandbinding surface for retinal. Whether E2 plays a related role in other rhodopsin-like GPCRs is unclear. To address this issue, we have now mutated to cysteine, one at a time, 10 consecutive residues in E2 of D2R. The reaction of five of these mutants with sulfhydryl reagents inhibited antagonist binding, and bound antagonist protected two, I184C and N186C, from reaction. The pattern of accessibility in E2 is consistent with a structure similar to that of bovine rhodopsin, in which the region C-terminal to the conserved disulfide bond is deeper in the binding-site crevice than is the N-terminal part of E2. Thus, E2 likely contributes to the binding site in the D2R and probably in other aminergic GPCRs as well. Knowledge of its detailed positioning and interactions with ligand would benefit GPCR molecular modeling and facilitate the design of novel drugs.T he extracellular loops are important in ligand binding in G protein-coupled receptors (GPCRs) with large molecular weight ligands, such as peptides (1). The role of these loops in aminergic GPCRs that bind small ligands has received much less attention, and it is widely believed that the transmembrane domain (TMD) is sufficient for ligand binding in these receptors (1). In the high-resolution bovine rhodopsin structure, however, the second extracellular loop (E2) folds down into the binding-site crevice to form a lid over retinal (2). It is unknown in aminergic GPCRs whether the E2 structure is similar to that of rhodopsin or whether E2 plays a role in ligand binding.For nearly all rhodopsin-like GPCRs, the disulfide bond between Cys 3.25 [Cys-107 in dopamine D 2 receptor (D2R)] and the conserved Cys in E2 (Cys e2, Cys-182 in D2R) connects E2 with the TMD § (Fig. 1A), and this disulfide bond (SS-E2) is crucial to the structural integrity and function of many GPCRs. The removal of SS-E2 by mutagenesis severely disrupts ligand binding to muscarinic acetylcholine receptors (3, 4) and destabilizes the high-affinity state of the ␤ 2 adrenergic receptor (AR) (5). Moreover, antagonist protected the ␤ 2 AR from the effects of reduction by DTT (6). Thus, SS-E2 is protected by a conformational change or steric block within the binding site.Although it had been argued that the presence of E2 within the TMD may be a feature unique to rhodopsin (7,8), several reports implicate E2 in ligand specificity in aminergic and other small molecule-ligand GPCRs (reviewed in ref. 9 and Discussion). Our studies of transmembrane segment 4 (TM4) of the D2R also suggested a role for the extracellular end of TM4 and the Nterminal part of E...

Accurate de novo structure prediction of large transmembrane protein domains using fragment-assembly and correlated mutation analysis

“…Interfacial helices were generally positioned correctly, for example in rhodopsin (1gzmA), where it is essential for binding the G-protein transducin suggesting that, in general, they form important stabilizing contacts with adjacent transmembrane helices or loops in addition to their expected role in constraining interhelix distances (41). Other than its N terminus, a series of beta strands that may form a "lid" over the retinal binding site (42) but that is poorly conserved across the whole family resulting in low contact prediction performance for this region, rhodopsin produces an excellent model with a TM-score of 0.65, with only minimal deviations from the native helix axes (Fig. 1A) and a TM-score of 0.79 over nonloop residues.…”

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

“…In a recent crystallographic study, a cytoplasmic domain directed nanobody binding induced subtle changes in binding pocket at Ͼ35 Å distance, and these changes led to active state orientation of critical serine residues that favor agonist binding (43). Induced conformational changes in the ECL2 have been documented in several GPCRs, including the D2 dopamine receptor and C5A complement receptor (44,45) upon ligand occupancy of the orthosteric pocket in the TM domain. Long range conformational changes associated with ligand binding have been shown in ECL1 of glucagon receptor (46); the N terminus, ECL1, and extracellular ends of all TM helices of Saccharomyces cerevisiae G protein-coupled receptor Ste2p are known (47)(48)(49).…”

Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

“…To compensate for the lower expression level of Ste2p-rho in LM102 in comparison to expression of the wild type Ste2p, we transformed pBKY1 into the BJS21 strain, which is deficient for proteases Pep4p, Prc1p and Prb1p. Pep4p encodes a vacuolar protease essential for proper maturation of many vacuolar enzymes [25] and thus is critical for the down-regulation and degradation of Ste2p in the vacuolar compartment [26]. Although the BJS21 strain is less sensitive to α-factor arrest due to the presence of the BAR1 gene, an extracellular protease that digests α-factor [27], we reasoned that, in a Pep4p deficient cell, the level of receptor expression would be increased compared to LM012 strain.…”