Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution.
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.
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
Matthias
M
Hillenbrand
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
Christian
C
Schori
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
Jendrik
J
Schöppe
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland plueckthun@bioc.uzh.ch.
Andreas
A
Plückthun
D000818:Animals; D002850:Chromatography, Gel; D020962:Heterotrimeric GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000302:isolation & purification / Q000378:metabolism*; D061987:Sf9 Cells; D012996:Solutions
D050505:Mutant Proteins; D043562:Receptors, G-Protein-Coupled; D012996:Solutions; D020962:Heterotrimeric GTP-Binding Proteins
G-protein–coupled receptor; heterotrimeric G proteins; membrane protein; protein complex; protein–protein interaction