Structure and function in rhodopsin: covalent crosslinking of the rhodopsin (metarhodopsin II)-transducin complex--the rhodopsin cytoplasmic face links to the transducin alpha subunit.

We prepared rhodopsin mutants that contained a single reactive cysteine residue per rhodopsin molecule at position 65, 140, 240, or 316 on the cytoplasmic face. A carbene-generating photoactivatable group was linked by a disulfide bond to the cysteine sulfhydryl group of each of the rhodopsin mutants. The resulting derivative was then light-activated at lambda > 495 nm to form the metarhodopsin II intermediate, which bound transducin. Subsequent photoactivation (355 nm) of the carbene-generating group resulted in crosslinking of the rhodopsin mutant carrying a cysteine residue at position 240 to transducin. This crosslinking was determined to be specifically with the alpha subunit of transducin. An alternative reaction observed during photolysis of the rhodopsin mutants was intramolecular insertion of the carbene into rhodopsin.

Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin.

Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.

Photoaffinity labeling the beta-adrenergic receptor with an iodoazido derivative of norepinephrine.

An iodinated photosensitive derivative of norepinephine, N-(p-azido-m-iodophenethylamidoisobutyl)-norepinephrine (NAIN), has been synthesized and characterized. NAIN stimulated adenylate cyclase activity in guinea pig lung membranes in a manner similar to (-)-isoproterenol and was inhibited by (-)-alprenolol. NAIN was shown to compete with [125I]iodocyanobenzylpindolol for the beta-adrenergic receptor in guinea pig lung membranes with an affinity which was dependent on the presence of guanyl nucleotides. Carrier-free radioiodinated NAIN ([125I]NAIN) was used at 2 nM to photoaffinity label the beta-adrenergic receptor in guinea pig lung membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of (-)-alprenolol (1 microM) protectable [125I]NAIN labeling showed the same molecular mass polypeptide (65 kDa) that was specifically derivatized with the antagonist photolabel [125I]iodoazidobenzylpindolol. Specific labeling of the beta-adrenergic receptor with [125I]NAIN was dependent on the presence of MgCl2 and the absence of guanyl nucleotide. Guanosine-5'-O-(3-thiotriphosphate (100 microM) abolished specific labeling by [125I]NAIN. N-Ethylmaleimide (2 mM) in the presence of [125I]NAIN protected against the magnesium and guanyl nucleotide effect. These data show that NAIN is an agonist photolabel for the beta-adrenergic receptor.