TM3 and the Conserved Cystine Bridge

The extracellular dithiol bridge between TM3 and the second outer loop is the only motif common to the 7TMR superfamily, apart from the ubiquitous presence of the

7TM helices. The second extracellular loop in rhodopsin produces a small region of antiparallel p-sheet, strands p3 and p4, which is extended by strands p1 and p2 from the amino terminal chain to produce a "plug" over retinal in the binding site (Figure 10.6). Retinal is located against TM3 and surrounded by a p-bonded cage of 12 aromatic residues, mainly tyrosine toward the solvent-exposed parts of the receptor and phenylalanine toward the center of the bilayer. The cyclohexenyl ring of retinal is wedged between Trp265648 and an aromatic stack supported by Cys167456. As noted above, the covalent attachment of retinal to Lys296743 is reinforced by the involvement of Cys264647, especially in fixing TM7 and in supporting the major p-bonded contribution from Trp265648. Cys264647 is also part of the retinal aromatic cage that isolates Lys296743.

The p4 strand arises from a helical turn that is divorced from the shortest helix 4, broken prematurely by a PP, or PxP, sequence in most receptors. The p4 strand is locked against TM3 by the cystine bond contributed by p4-Cys187 and the conserved cysteine at the amino terminal end of TM3, CysHO3 25. The p4 strand is H-bonded to the retinal polyene chain, almost as if it were an extension of the p sheet (Figure 10.6A). The integrity of the extensive aromatic sulfur cage is critically dependent upon the juxtaposition of residues imposed by the restraining cystine bond between TM3 and p4.

From the earliest cloning days, SDM of all of the individual cysteines in the p2-AR demonstrated that those in the second extracellular loop were important for agonist binding,44 rationalizing the many biochemical reports showing that reduction generally increased receptor-mediated activity, and usually at the expense of receptor stability. These original observations were confirmed by innumerable reports using the same and other 7TMRs and almost invariably attributed to an indispensable structural role for the extracellular dithiol bridge (Figure 10.7).

FLEXIBILITY

FIGURE 10.7 Flexibility and redox provide a broad range of binding and activity modalities. Membrane receptors sample the external oxidizing and internal reducing environments of the cell and are mediators of signal transduction between external ligands and internal second messenger systems, possibly by exploiting the redox potential gradient across the membrane.

structural cysteine

SIGNAL TRANSDUCTION

FLEXIBILITY

'active' cysteine

FIGURE 10.7 Flexibility and redox provide a broad range of binding and activity modalities. Membrane receptors sample the external oxidizing and internal reducing environments of the cell and are mediators of signal transduction between external ligands and internal second messenger systems, possibly by exploiting the redox potential gradient across the membrane.

The receptor sits in the plasma membrane and samples the external oxidizing and intracellular reducing environments simultaneously. Extracellular cysteines are presumed to be relatively inert structural cysteines forming molecular bridges, whereas intracellular cysteines are highly reactive and often enzymatically active. For a comprehensive overview of the chemical activities of cysteines and involvement in cellular processes, see the excellent reviews from Jacob and co-authors.45,46

Having a disulfide bridge that is structural does not preclude the same bridge from involvement in the functional activity of the receptor. We considered that signal transduction induced by ligand binding could involve the exterior disulfide-bonded cysteine residues interacting, directly or indirectly, with the reduced cysteine residues of the G protein cascade within the cell. This process can be modeled as a progressive extracellular-to-intracellular disulfide-thiol exchange initiated by external ligand binding that transduces the signal internally.

This proposition satisfied a number of prejudices and expectations: (1) reduction of the cystine bridge may be the liberating event for activation and allosteric or conformational changes in the receptor; (2) flexibility was already identified as a major characteristic of multispanning TMRs; (3) the two together would provide a broad range of binding and activation modalities, sufficient to accommodate such a diverse set of ligands, temporal responses, and second messenger specificities; (4) reduction of the cystine bridge would also liberate the participating cysteines for labeling by thiol-reactive reagents; and (5) what better way to communicate a signal from the outer oxidizing environment to the inner reducing environment than by the invocation of a redox-dependent switch?

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