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PMID: 25733868 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 112 ·No. 11 ·2015-03-17 ·Pages E1181-90

Hillenbrand M, Schori C, Schöppe J, Plückthun A

Abstract

Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.

Keywords
G-protein–coupled receptor heterotrimeric G proteins membrane protein protein complex protein–protein interaction
MeSH Terms
Animals Chromatography, Gel Heterotrimeric GTP-Binding Proteins/isolation & purification,metabolism Humans Mutant Proteins/metabolism Protein Binding Receptors, G-Protein-Coupled/isolation & purification,metabolism Sf9 Cells Solutions
Chemicals
Mutant Proteins Receptors, G-Protein-Coupled Solutions Heterotrimeric GTP-Binding Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Hillenbrand Matthias
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
Schori Christian
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
Schöppe Jendrik
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
Plückthun Andreas
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland plueckthun@bioc.uzh.ch.
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
1091-6490
DOI
10.1073/pnas.1417573112
Email
plueckthun@bioc.uzh.ch
Published
2015-03-17
Epub
2015-00-02
Pages
E1181-90
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC4371982
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.1417573112 PMC Full Text View journal details
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