Biology subjects

Dwivedi-Agnihotri, H.

Publications and source records attributed to Dwivedi-Agnihotri, H..

3 recordsLinked to original sources

bioRxiv2021

Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

G protein-coupled receptors (GPCRs) are typically characterized by their seven transmembrane (7TM) architecture, and interaction with two universal signal-transducers namely, the heterotrimeric G-proteins and {beta}-arrestins ({beta}arrs). Synthetic ligands and receptor mutants have been designed to elicit transducer-coupling preferences and distinct downstream signaling outcomes for many GPCRs. This raises the question if some naturally-occurring 7TMRs may selectively engage one of these two signal-transducers, even in response to their endogenous agonists. Although there are scattered hints in the literature that some 7TMRs lack G-protein coupling but interact with {beta}arrs, an in-depth understanding of their transducer-coupling preference, GRK-engagement, downstream signaling and structural mechanism remains elusive. Here, we use an array of cellular, biochemical and structural approaches to comprehensively characterize two non-canonical 7TMRs namely, the human decoy D6 receptor (D6R) and the human complement C5a receptor (C5aR2), in parallel with their canonical GPCR counterparts, CCR2 and C5aR1, respectively. We discover that D6R and C5aR2 couple exclusively to {beta}arrs, exhibit distinct GRK-preference, and activate non-canonical downstream signaling partners. We also observe that {beta}arrs, in complex with these receptors, adopt distinct conformations compared to their canonical GPCR counterparts despite being activated by a common natural agonist. Our study therefore establishes D6R and C5aR2 as bona-fide arrestin-coupled receptors (ACRs), and provides important insights into their regulation by GRKs and downstream signaling with direct implications for biased agonism.

biochemistry↗

bioRxiv2020

Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor

The {beta}1-adrenoceptor ({beta}1AR) is a G protein-coupled receptor (GPCR) activated by the hormone noradrenaline, resulting in the coupling of the heterotrimeric G protein Gs1. G protein-mediated signalling is terminated by phosphorylation of the receptor C-terminus and coupling of {beta}-arrestin 1 ({beta}arr1, also known as arrestin-2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins (biased agonism)3 is important in drug development, as the therapeutic effect may arise from only one signalling cascade, whilst the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, we determined the electron cryo-microscopy (cryo-EM) structure of the {beta}1AR-{beta}arr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound {beta}1AR coupled to the G protein mimetic nanobody Nb806. {beta}arr1 couples to {beta}1AR in a distinct manner to how Gs couples to {beta}2AR7, with the finger loop of {beta}arr1 occupying a narrower cleft on the intracellular surface closer to transmembrane helix H7 than the C-terminal 5 helix of Gs. The conformation of the finger loop in {beta}arr1 is different from that adopted by the finger loop in visual arrestin when it couples to rhodopsin8, and its {beta}-turn configuration is reminiscent of the loop in Nb80 that inserts at the same position. {beta}1AR coupled to {beta}arr1 showed significant differences in structure compared to {beta}1AR coupled to Nb80, including an inward movement of extracellular loop 3 (ECL3) and the cytoplasmic ends of H5 and H6. In the orthosteric binding site there was also weakening of interactions between formoterol and the residues Ser2115.42 and Ser2155.46, and a reduction in affinity of formoterol for the {beta}1AR-{beta}arr1 complex compared to {beta}1AR coupled to mini-Gs. These differences provide a foundation for the development of small molecules that could bias signalling in the {beta}-adrenoceptors.

molecular biology↗

bioRxiv2019

Purification of native CCL7 and its functional interaction with selected chemokine receptors

Chemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its 15N-, 15N/13C-, 2H/15N/13C-isotope-labeled derivatives, at milligram levels using E. coli expression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and 1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and {beta}-arrestins for selected chemokine receptors in cellular system. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies.

biochemistry↗