Functional characterization of rhodopsin monomers and dimers in detergents.
Rhodopsin (Rho) is a G protein-coupled receptor that initiates phototransduction in rod photoreceptors. High expression levels of Rho in the disc membranes of rod outer segments and the propensity of Rho to form higher oligomeric structures are evident from atomic force microscopy, transmission electron microscopy, and chemical cross-linking experiments. To explore the structural and functional properties of Rho in n-dodecyl-beta-maltoside, frequently used to purify heterologously expressed Rho and its mutants, we used gel filtration techniques, blue native gel electrophoresis, and functional assays. Here, we show that in micelles containing n-dodecyl-beta-maltoside at concentrations greater than 3 mM, Rho is present as a single monomer per detergent micelle. In contrast, in 12 mM 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), micelles contain mostly dimeric Rho. The cognate G protein transducin (Gt) appears to have a preference for binding to the Rho dimer, and the complexes fall apart in the presence of guanosine 5'-3-O-(thio)triphosphate. Cross-linked Rho dimers release the chromophore at a slower rate than monomers and are much more resistant to heat denaturation. Both Rho(*) monomers and dimers are capable of activating Gt, and both of them are phosphorylated by Rho kinase. Rho expressed in HEK293 cells is also readily cross-linked by a bifunctional reagent. These studies provide an explanation of how detergent influences the oligomer-dimermonomer equilibrium of Rho and describe the functional characterization of Rho monomers and dimers in detergent.
Department of Ophthalmology, University of Washington, Seattle, Washington 98195, USA.
Beata
B
Jastrzebska
Tadao
T
Maeda
Li
L
Zhu
Dimitrios
D
Fotiadis
Slawomir
S
Filipek
Andreas
A
Engel
Ronald E
RE
Stenkamp
Krzysztof
K
Palczewski
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002460:Cell Line; D002793:Cholic Acids / Q000737:chemistry; D003432:Cross-Linking Reagents; D003460:Crystallization; D003902:Detergents / Q000737:chemistry*; D019281:Dimerization; D015870:Gene Expression; D005960:Glucosides / Q000737:chemistry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006358:Hot Temperature; D047908:Intracellular Signaling Peptides and Proteins; D008823:Micelles; D008958:Models, Molecular; D015394:Molecular Structure; D010766:Phosphorylation; D011489:Protein Denaturation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D012374:Rod Cell Outer Segment / Q000737:chemistry; D013329:Structure-Activity Relationship; D013388:Succinimides; D015293:Transducin / Q000378:metabolism; D054460:rho-Associated Kinases
D000911:Antibodies, Monoclonal; D002793:Cholic Acids; D003432:Cross-Linking Reagents; D003902:Detergents; D005960:Glucosides; D047908:Intracellular Signaling Peptides and Proteins; D008823:Micelles; D013388:Succinimides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C040358:dodecyl maltoside; D012243:Rhodopsin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D015293:Transducin; C011240:dithiobis(succinimidylpropionate); C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate