A surprising feature of bovine rhodopsin is the highly structured extracellular N terminus and extracellular loops . In particular, the second extracellular loop (E2), which connects TM4 and TM5, dives down into the transmembrane domain and forms a "plug" that contacts retinal (Table 1 and Fig. 2). This loop also contains a highly conserved Cys that is disulfide-bonded to another highly conserved Cys at the top of TM3 . E2 contains two stretches of P-strand, one of which, P4, lies directly over retinal . E2 thus forms a lid over retinal and protects it from the extracellular milieu. Given the high degree of conservation of the amino acids in the P4 strand in vertebrate opsins, and the variability within this region in other class A receptors, the prevailing view was that the P4 strand might serve specifically to define the retinal-binding pocket in vertebrate opsins and not other GPCRs [12,13]. We suggest that this response is at least partly wrong, for a number of reasons discussed below.
We do not yet know of any structural similarity of E2 between rhodopsin and other class A receptors beyond the shared disulfide bond, but the sequence at the extracellular end of TM4 and the beginning of E2 is highly conserved among functionally related receptors and among species variants of these receptors, despite the fact that the sequence of E2 is highly variable across class A receptors . In addition, this region has been identified as the site of cova-lent attachment of photoaffinity derivatives of agonist and antagonist ligands of the a2-AR , and mutations in this region have ligand-specific effects (reviewed in Javitch et al. ). Moreover, known ligand binding sites in other TMs are predicted to be in spatial proximity to this region. It is likely, therefore, that this region plays a functional role, and it
Figure 3 Ligand binding crevice. In (A), the residues in the TMs that were identified from the SASA analysis (see text, Table 1, and Fig. 2) are shown in van der Waals representation, with retinal bound within the surface created by these residues. In (B), the side chains of residues from the P2-adrenergic receptor are shown on the backbone of the rhodopsin structure. These residues have been experimentally determined to interact with catecholamine ligands, and include Asp113332, Ser203542, Ser204543, Ser207546, Phe208547, Trp286648, Phe289651, Phe290652, and Asn293655, a subset of the positions shown in (A). In (C), IpBABC (p- (bromoacetamido) benzyl-1-[125I]iodocarazolol), an affinity label derivative of pindodol, is docked within the TMs of the rhodopsin structure with the TM residues from Table 1 mutated to the aligned P2-adrenergic receptor residues. IpBABC is shown covalently attached to His932 64 in TM2. Residues with the same index number are shown in the same color in all three panels. The residues displayed next to each other are shown in different colors. (From Ballesteros, J. et al., Mol. Pharmacol. 60, 1-19, 2001. With permission.) A color representation of this figure is available on the CD version of the Handbook of Cell Signaling.
is possible that an orientation of E2 similar to that in rhodopsin may explain these findings.
Several reports implicate E2 in ligand specificity in a number of small molecule-ligand GPCRs. Perez and colleagues found that substitution of three consecutive residues in E2 changed the ligand specificity for particular antagonists from that of a1BAR to that of a1AAR, and vice versa . Similarly, substitution of E2 and TM5 altered the subtype specificity of the 5-HT1D receptor to that of the 5-HT1B receptor and vice versa , and substitution of a single residue in E2 was also sufficient to interconvert the pharmacological specificity of canine 5-HT1D and human 5-HT1D receptor . In adenosine receptor, in which the binding site is also formed in the transmembrane domain , several glutamate residues in E2 are critical for ligand recognition [20,21]. Although it is currently difficult to envision the entrance route of ligands into the binding-site crevice and the potential associated conformational rearrangements of E2, these data nonetheless suggest a direct role of residues in E2 in ligand binding in other class A receptors .
We are grateful to all our current and former colleagues and collaborators, and especially to Myles Akabas, Juan Ballesteros, Arthur Karlin, and Harel Weinstein for much helpful discussion, and to NIMH grants 57324 and 54137, the Lebovitz Foundation, and the Lieber Center for support.
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