Cholesterol dependent recruitment of di22:6-PC by a G protein-coupled receptor into lateral domains.
Bovine rhodopsin was reconstituted into mixtures of didocosahexaenoylphosphatidylcholine (di22:6-PC), dipalmitoylphosphatidylcholine (di16:0-PC), sn-1-palmitoyl-sn-2-docosahexaenoylphosphatidylcholine (16:0, 22:6-PC) and cholesterol. Rhodopsin denaturation was examined by using high-sensitivity differential scanning calorimetry. The unfolding temperature was increased at lower levels of lipid unsaturation, but the highest temperature was detected for native disk membranes: di22:6-PC < 16:0,22:6-PC < di16:0,18:1-PC < native disks. The incorporation of 30 mol% of cholesterol resulted in 2-4 degrees C increase of denaturation temperature in all reconstituted systems examined. From the analysis of van't Hoff's and calorimetric enthalpies, it was concluded that the presence of cholesterol in di22:6-PC-containing bilayers induces a level of cooperativity in rhodopsin unfolding. Fluorescence resonance energy transfer (FRET), using lipids labeled at the headgroup with pyrene (Py) as donors and rhodopsin retinal group as acceptor of fluorescence, was used to study rhodopsin association with lipids. Higher FRET efficiencies detected for di22:6-PE-Py, compared to di16:0-PE-Py, in mixed di22:6-PC-di16:0-PC-cholesterol bilayers, indicate preferential segregation of rhodopsin with polyunsaturated lipids. The effective range of the rhodopsin-lipid interactions facilitating cluster formation exceeds two adjacent lipid layers. In similar mixed bilayers containing no cholesterol, cluster formation was absent at temperatures above lipid phase transition, indicating a crucial role of cholesterol in microdomain formation.
Polozova
A
A
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland 20852, USA.
Litman
B J
BJ
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism*; D004355:Drug Stability; D004735:Energy Transfer; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D015335:Molecular Probes; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D011721:Pyrenes / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism
D008563:Membrane Lipids; D015335:Molecular Probes; D010713:Phosphatidylcholines; D011721:Pyrenes; D011956:Receptors, Cell Surface; C417741:didocosahexaenoylphosphatidylcholine; D012243:Rhodopsin; D002784:Cholesterol; D019204:GTP-Binding Proteins