RT Journal Article
SR Electronic
T1 Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.01.11.475811
DO 10.1101/2022.01.11.475811
A1 Mithu Baidya
A1 Madhu Chaturvedi
A1 Hemlata Dwivedi-Agnihotri
A1 Ashutosh Ranjan
A1 Dominic Devost
A1 Yoon Namkung
A1 Tomasz Maciej Stepniewski
A1 Shubhi Pandey
A1 Minakshi Baruah
A1 Bhanupriya Panigrahi
A1 Jagannath Maharana
A1 Ramanuj Banerjee
A1 Jana Selent
A1 Stephane Laporte
A1 Terence E. Hebert
A1 Arun K. Shukla
YR 2022
UL http://biorxiv.org/content/early/2022/01/11/2022.01.11.475811.abstract
AB Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for βarrs, agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of βarr translocation to endosomes although βarrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like βarr1 conformation, rescues endosomal translocation of βarr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in βarr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of βarr1 with β2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of βarr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery.Significance The interaction of G protein-coupled receptors (GPCRs) with β-arrestins (βarrs) is a critical step in their regulatory and signaling paradigms. While intrabodies that bind to GPCRs, G proteins and βarrs have been utilized as biosensors and regulators of functional outcomes, allosteric targeting of receptor-transducer complexes to encode gain of function has not been documented so far. Here, we discover that a conformation-specific synthetic intrabody recognizing GPCR-bound βarr1 can allosterically enhance endosomal trafficking of βarr1 and agonist-induced ERK1/2 MAP kinase activation. This intrabody promotes an active-like βarr1 conformation and enhances the interaction of β2-adaptin with βarr1. Our findings establish a conceptual framework to allosterically modulate protein-protein interactions in GPCR signaling cascade to modulate their trafficking and signaling responses.Competing Interest StatementThe authors have declared no competing interest.