Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction

doi:10.1073/pnas.0705312104

Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction

*Membrane Protein Structure and Function Unit, National Institute of Neurological Disorders and Stroke, and
^‡Laboratory of Chemical Physics and
^§Biotechnology Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892; and
^¶Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Department of Health and Human Services, Rockville, MD 20850

Communicated by Wayne A. Hendrickson, Columbia University, New York, NY, June 6, 2007 (received for review December 21, 2006)

Abstract

G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concentrations, NTS1 binds the agonist neurotensin with a Hill slope of ≈1; at higher receptor concentrations, neurotensin binding displays positive cooperativity with a Hill slope of ≈2. NTS1 monomers activate Gαqβ₁γ₂, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.

Footnotes

^‖To whom correspondence should be addressed at:
NINDS, NIH, 5625 Fishers Lane, Room 4S-12, Rockville, MD 20852.
E-mail: rkgriss{at}helix.nih.gov
Author contributions: J.F.W., J. Grodnitzky, J.K.N., and R.G. designed research; J.F.W., J. Grodnitzky, J.M.L., L.B.T., J.S., J. Gutierrez, J.K.N., and R.G. performed research; J.F.W., J. Grodnitzky, J.M.L., J.K.N., and R.G. analyzed data; and J.K.N. and R.G. wrote the paper.
↵ ^†Present address: Department of Biomedical Sciences, Iowa State University, Ames, IA 50011.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0705312104/DC1.
Abbreviations:

GPCR,

G protein-coupled receptor;

NT,

neurotensin;

SEC,

size exclusion chromatography;

LS,

light scattering;

RI,

refractive index;

GTPγS,

guanosine 5′-[γ-thio]triphosphate;

LM,

n-dodecyl-β-d-maltoside;

CHS,

cholesteryl hemisuccinate Tris salt.
Freely available online through the PNAS open access option.