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PMID: 11053136 Published · ppublish English Journal Article

Cholesterol dependent recruitment of di22:6-PC by a G protein-coupled receptor into lateral domains.

Biophysical journal ·Vol. 79 ·No. 5 ·2000-11-00 ·Pages 2632-43

Polozova A, Litman BJ

Abstract

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.

MeSH Terms
Animals Biophysical Phenomena Biophysics Cattle Cholesterol/metabolism Drug Stability Energy Transfer GTP-Binding Proteins/metabolism In Vitro Techniques Membrane Lipids/chemistry,metabolism Molecular Probes Phosphatidylcholines/chemistry,metabolism Protein Denaturation Pyrenes/chemistry Receptors, Cell Surface/metabolism Rhodopsin/metabolism
Chemicals
Membrane Lipids Molecular Probes Phosphatidylcholines Pyrenes Receptors, Cell Surface didocosahexaenoylphosphatidylcholine Rhodopsin Cholesterol GTP-Binding Proteins
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Polozova 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
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Article Info
Journal
Biophysical journal
Abbr.
Biophys J
ISSN
0006-3495
DOI
10.1016/S0006-3495(00)76502-7
Published
2000-11-00
Pages
2632-43
Language
English
Region
United States
NLM ID
0370626
PMCID
PMC1301144
Subset
IM
External Links
PubMed source DOI: 10.1016/S0006-3495(00)76502-7 PMC Full Text View journal details
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