Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.
Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.
Harding
Peter J
PJ
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
Attrill
Helen
H
Boehringer
Jonas
J
Ross
Simon
S
Wadhams
George H
GH
Smith
Eleanor
E
Armitage
Judith P
JP
Watts
Anthony
A
D000595:Amino Acid Sequence; D001921:Brain / Q000166:cytology / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism*; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D011510:Proteolipids / Q000378:metabolism; D018028:Receptors, Neurotensin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling; D013379:Substrate Specificity
C485182:Cyan Fluorescent Protein; D003902:Detergents; D008051:Lipid Bilayers; D008081:Liposomes; D010743:Phospholipids; D011510:Proteolipids; D018028:Receptors, Neurotensin; C114036:neurotensin type 1 receptor; C020485:proteoliposomes; D049452:Green Fluorescent Proteins