The Effects of Amino Acid Replacements of Glycine 121 on Transmembrane Helix 3 of Rhodopsin

May Han,Steven W Lin,Steven O Smith,Thomas P Sakmar

doi:10.1074/jbc.271.50.32330

May Han, Steven W Lin + Show 2 more

Open Access

https://doi.org/10.1074/jbc.271.50.32330

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Abstract

Rhodopsin is a member of a family of G protein-coupled receptors with seven transmembrane (TM) helices. In rhodopsin, Gly121 is a highly conserved amino acid residue near the middle of TM helix 3. TM helix 3 is known to be involved in chromophore-protein interactions and contains the chromophore Schiff base counterion at position 113. We prepared a set of seven single amino acid replacement mutants of rhodopsin at position 121 (G121A, Ser, Thr, Val, Ile, Leu, and Trp) and control mutants with replacements of Gly114 or Ala117. The mutant opsins were expressed in COS cells and reconstituted with either 11-cis-retinal, the ground-state chromophore of rhodopsin, or all-trans-retinal, the isomer formed upon receptor photoactivation. The replacement of Gly121 resulted in a relative reversal in the selectivity of the opsin apoprotein for reconstitution with 11-cis-retinal over all-trans-retinal in COS cell membranes. The mutant pigments also were found to be thermally unstable to varying degrees and reactive to hydroxylamine in the dark. In addition, the size of the residue substituted at position 121 correlated directly to the degree of blue-shift in the lambdamax value of the pigment. These results suggest that Gly121 is an important and specific component of the 11-cis-retinal binding pocket in rhodopsin.

Highlights

Rhodopsin is a member of a family of G protein-coupled receptors with seven transmembrane (TM) helices
In the most recent model, NMR constraints were used to position the chromophore in the interior of rhodopsin, which resulted in retinal situated between TM helices 3 and 6 with the ␤-ionone ring oriented toward TM helix 5 [19, 20]
Spectral Properties of TM Helix 3 Mutants—Mutant opsin genes were prepared with single amino acid replacements at one of three sites in TM helix 3 of bovine rhodopsin, Gly114, Ala117, and Gly121 (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Materials—Sources of reagents and materials have been previously reported [4, 15, 23, 24]. For pigment samples purified in dodecyl maltoside, pigment concentration was determined before the assay by visible spectroscopy according to the ⑀ values given in Tables I and II. The activity assays of mutant opsins in COS cell membranes were performed as follows. Regeneration Kinetics of Mutant Opsin G121L with Retinals in Membranes—The regeneration time courses of mutant G121L with 11-cisretinal or all-trans-retinal in COS cell membranes was measured by monitoring the initial rate of transducin activation at various periods after the addition of retinal to membrane aliquots. 11-cis or all-trans (2 ␮l of 25 ␮M ethanolic solution), was mixed with 60 ␮l of COS cell membrane suspension containing mutant opsin G121L in darkness and incubated at room temperature. The spectral properties of the mutant pigments, including ␭max values, are presented in Table I. filter-binding method to determine transducin activity. The experimental data were fitted by an exponential-rise function of the form, y ϭ a (1 Ϫ exp(Ϫbx)) ϩ c

RESULTS

Spectral ratiob

Spectral ratio

DISCUSSION