TY - JOUR T1 - Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody JF - bioRxiv DO - 10.1101/2022.01.11.475811 SP - 2022.01.11.475811 AU - Mithu Baidya AU - Madhu Chaturvedi AU - Hemlata Dwivedi-Agnihotri AU - Ashutosh Ranjan AU - Dominic Devost AU - Yoon Namkung AU - Tomasz Maciej Stepniewski AU - Shubhi Pandey AU - Minakshi Baruah AU - Bhanupriya Panigrahi AU - Jagannath Maharana AU - Ramanuj Banerjee AU - Jana Selent AU - Stephane Laporte AU - Terence E. Hebert AU - Arun K. Shukla Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/01/11/2022.01.11.475811.abstract N2 - 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. ER -