Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor

Nature Communications
Sahil GulatiKrzysztof Palczewski

Abstract

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by mediating a GDP to GTP exchange in the Gα subunit. This leads to dissociation of the heterotrimer into Gα-GTP and Gβγ dimer. The Gα-GTP and Gβγ dimer each regulate a variety of downstream pathways to control various aspects of human physiology. Dysregulated Gβγ-signaling is a central element of various neurological and cancer-related anomalies. However, Gβγ also serves as a negative regulator of Gα that is essential for G protein inactivation, and thus has the potential for numerous side effects when targeted therapeutically. Here we report a llama-derived nanobody (Nb5) that binds tightly to the Gβγ dimer. Nb5 responds to all combinations of β-subtypes and γ-subtypes and competes with other Gβγ-regulatory proteins for a common binding site on the Gβγ dimer. Despite its inhibitory effect on Gβγ-mediated signaling, Nb5 has no effect on Gαq-mediated and Gαs-mediated signaling events in living cells.

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Datasets Mentioned

BETA
AF501883
M31328
AF300648
PXD009503

Methods Mentioned

BETA
ELISA
pull down
surface plasmon resonance
affinity purification
BRET
bioluminescence resonance
transfection
x-ray crystallography
phage display
size exclusion chromatography

Software Mentioned

Xcalibur
Qual Browser
wwPDB
CCP4
MassMatrix
PHASER
HXExpress
PISA
Coot
ProteomeXchange

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