Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.

Twenty-one single-cysteine substitution mutants were prepared in the sequence 56-75 between transmembrane helices I and II at the cytoplasmic surface of bovine rhodopsin. Each mutant was reacted with a sulfhydryl-specific reagent to produce a nitroxide side chain. The electron paramagnetic resonance of the labeled proteins in dodecyl maltoside solution was analyzed to provide the relative mobility and accessibility of the nitroxide side chain to both polar and nonpolar paramagnetic reagents. The results indicate that the hydrophobic-water interface of the micelle intersects helices I and II near residues 64 and 71, respectively. Thus, the sequence 64-71 is in the aqueous phase, while 56-63 and 72-75 lie in the transmembrane helices I and II, respectively. The lipid-facing surfaces on transmembrane helices I and II near the cytoplasmic surface correspond to approximately 180 degrees and 90 degrees of arc on the helical surfaces, respectively. Photoactivation of rhodopsin produced changes in structure in the region investigated, primarily around helix II. However, these changes are much smaller than those noted by spin labels in helix VI (Altenbach, C., Yang, K., Farrens, D., Farahbakhsh, Z., Khorana, H. G., and Hubbell, W. L. (1996) Biochemistry 35, 12470).

Cysteines were introduced, one at a time, at amino acid positions 55-75 in the cytoplasmic region connecting helices I and II in rhodopsin. In each of the 21 cysteine mutants, the reactive native cysteine residues (C140 and C316) were replaced by serine. Except for N55C, all mutants formed rhodopsin-like chromophores and had normal photobleaching characteristics. The efficiency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The reactivity of the substituted cysteine in each mutant toward 4, 4'-dithiodipyridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and I75C were unreactive to 4-PDS under the conditions used, suggesting that they are embedded in the micelle or protein interior. The mutants V63C, H65C-T70C, and N73C reacted rapidly, while the remainder of the mutants reacted more slowly, and varied in reactivity with sequence position. For the mutants derivatized with 4-PDS, the rate of release of thiopyridone from the resulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investigated in the dark and in the light. Marked changes in the rates of thiopyridone release in the light were found at specific sites. Collectively, the data reveal tertiary interactions of the residues in the sequence investigated and demonstrate structural changes due to photoactivation.

Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end of helix III) and Cys250 (end of helix VI); disulfide bond 5, between Cys135 (end of helix III) and Cys250 (end of helix VI); and disulfide bond 6, between Cys245 (end of helix VI) and Cys338 (C-terminus). The effects of local restrictions caused by the cross-links on transducin (G(T)) activation and phosphorylation by rhodopsin kinase (RK) following illumination were studied. Disulfide bond 1 showed little effect on either G(T) activation or phosphorylation by RK, suggesting that the relative motion between interhelical loop I-II and helix VII is not crucial for recognition by G(T) or by RK. In contrast, disulfide bonds 2-5 abolished both G(T) activation and phosphorylation by RK. Disulfide bond 6 resulted in enhanced G(T) activation but abolished phosphorylation by RK, suggesting the structure recognized by G(T) was stabilized in this mutant by cross-linking of the C-terminus to the cytoplasmic end of helix VI. Thus, the consequences of the disulfide cross-links depended on the location of the restriction. In particular, relative motions of helix VI, with respect to both helices III and VII upon light activation, are required for recognition of rhodopsin by both G(T) and RK. Further, the conformational changes in the cytoplasmic face that are necessary for protein-protein interactions need not be cooperative, and may be segmental.