Biology subjects

Chandan, N. R.

Publications and source records attributed to Chandan, N. R..

3 recordsLinked to original sources

bioRxiv2025

GPR97/ADGRG3 is activated by its tethered peptide agonist and not by steroids to induce neutrophil polarization and migration

Most adhesion G protein coupled receptors (AGPCRs) are activated by intramolecular binding of a tethered-peptide agonist (TA). Shear force-induced dissociation of the AGPCR N-terminal fragment (NTF) and C-terminal fragment (CTF) exposes the TA. The decrypted TA binds rapidly to its orthosteric site within the CTF to stabilize the active state of the AGPCR. Corticosteroids were previously proposed to be agonists for GPR97/ADGRG3. Later, other steroids and androgens were purported to be selective agonists of additional AGPCRs. Here, we demonstrate that GPR97/ADGRG3 is activated by dissociation of its NTF/CTF and follows the TA mechanism. TA peptidomimetics and the ADGRG subfamily partial agonist 3-acetoxydihydrodeoxygeduin (3--DOG), but not corticoids, stimulated GPR97/ADGRG3 in cell-based luciferase reporter assays and receptor/G protein reconstitution assays. GPR97 was defined as a promiscuous AGPCR that couples to G13, Gs and Gi, but not Gq. GPR97 is highly expressed in human polymorphonuclear neutrophils (hPMNs). Neutrophils undergo actin polymerization-induced cell shape changes and polarization and migrate upon activation. We found that GPR97 activation via TA peptidomimetics or 3--DOG robustly stimulated hPMN and mouse bone-marrow neutrophil (mBMN) polarization. Furthermore, GPR97 TA peptidomimetics and 3--DOG, but not beclomethasone, induced hPMN and mBMN chemotaxis. Together, our results demonstrate that GPR97/ADGRG3 utilizes a tethered agonist mechanism to activate G protein signaling and induce neutrophil polarization and migration. One Sentence SummaryGPR97/ADGRG3 tethered agonism regulates G protein signaling in neutrophils.

pharmacology and toxicology↗

bioRxiv2023

An Ionic Lock Mediates Interdomain Interactions in G protein αi subunits and Determines Gαi Subtype Signaling Specificity

Highly homologous members of the Gi family, Gi1-3, have distinct tissue distributions and physiological functions, yet the functional properties of these proteins with respect to GDP/GTP binding and regulation of adenylate cyclase are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gi1 effector, however, it is poorly activated by Gi2. Here, in a proteomic proximity labeling screen we observed a strong preference for Gi1 relative to Gi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gi1 into Gi2 or substitution of a single amino acid, A230 in Gi2 to the corresponding D in Gi1, largely rescues PRG activation and interactions with other Gi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, dynamic separation at the HD-Ras-like domain (RLD) interface is prevalent in Gi2 relative to Gi1 and that mutation of A230s4h3.3 to D in Gi2 stabilizes HD-RLD interactions through formation of an ionic interaction with R145HD.11 in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229s4h3.3 in Gi1 interacts with R144HD.11 stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gi2 is unable to form the "ionic lock" to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gi1 or Gi2 by GPCRs.

pharmacology and toxicology↗

bioRxiv2021

Identification of G Protein αi Signaling Partners by Proximity Labeling Reveals a Network of Interactions that Includes PDZ-RhoGEF

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gi in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gi in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gi1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gi1(Q204L), suggesting a previously unappreciated network of interactions for activated Gi proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gi1 strongly activates PRG. Strikingly, large differences in the ability of Gi1, Gi2, and Gi3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gi and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gi both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches. SummaryProximity-based labeling approach was used to identify signaling networks and signaling mechanisms downstream of Gi-coupled receptors.

pharmacology and toxicology↗