Biology subjects

Dwivedi, H.

Publications and source records attributed to Dwivedi, H..

4 recordsLinked to original sources

bioRxiv2019

Crystal structure of β-arrestin 2 in complex with an atypical chemokine receptor phosphopeptide reveals an alternative active conformation

{beta}-arrestins ({beta}arrs) critically regulate signaling and trafficking of G protein-coupled receptors (GPCRs), the largest family of drug targets in the human genome, and there are two isoforms of {beta}arrs: {beta}arr1 and {beta}arr2. Most GPCRs interact with both the heterotrimeric G-proteins and {beta}arrs, inducing distinct downstream signal transduction. However, certain chemokine receptors lack functional G-protein coupling, but they can efficiently recruit {beta}arrs upon agonist-stimulation, and they are referred to as atypical chemokine receptors (ACKRs). Receptor phosphorylation is a key determinant for the binding of {beta}arrs, and understanding the intricate details of receptor-{beta}arr interaction is the next frontier in GPCR structural biology. To date, the high-resolution structures of active {beta}arr1 have been revealed, but the activation mechanism of {beta}arr2 by a phosphorylated GPCR remains elusive. Here, we present a 1.95 [A] crystal structure of {beta}arr2 in complex with a phosphopeptide (C7pp) derived from the carboxyl-terminus of ACKR3, also known as CXCR7. The structure of C7pp-bound {beta}arr2 reveals key differences from the previously determined active conformation of {beta}arr1. One of the key differences is that C7pp-bound {beta}arr2 shows a relatively small inter-domain rotation. An antibody-fragment-based conformational sensor and hydrogen/deuterium exchange experiments further corroborate structural features and suggest that the determined structure is an alternative active conformation of {beta}arr2.

biochemistry↗

bioRxiv2019

Conformational sensors and domain-swapping reveal structural and functional differences between β-arrestin isoforms

Desensitization, signaling and trafficking of G protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, {beta}-arrestins ({beta}arrs). The two isoforms of {beta}arrs ({beta}arr1 and 2) share a high degree of sequence and structural similarity, still however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of {beta}arr isoforms is still lacking. Using a set of complementary approaches including antibody fragment based conformational sensors, we discover structural differences between {beta}arr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain swapped chimeras of {beta}arrs display robust complementation in functional assays thereby, linking the structural differences between the receptor-bound {beta}arr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of {beta}arr isoforms to drive distinct functional outcomes, and underscore the importance of integrating this aspect in the current framework of biased agonism.

biophysics↗

bioRxiv2019

Genetically encoded intrabody sensors illuminate structural and functional diversity in GPCR-β-arrestin complexes

Interaction of {beta}-arrestins ({beta}arrs) upon agonist-stimulation is a hallmark of G protein-coupled receptors (GPCRs) resulting in receptor desensitization, endocytosis and signaling. Although overall functional roles of {beta}arrs are typically believed to be conserved across different receptors, emerging data now clearly unveils receptor-specific functional contribution of {beta}arrs. The underlying mechanism however remains mostly speculative and represents a key missing link in our current understanding of GPCR signaling and regulatory paradigms. Here, we develop synthetic intrabody-based conformational sensors that help us visualize the assembly and trafficking of GPCR-{beta}arr1 complexes in cellular context for a broad set of receptors with spatio-temporal resolution. Surprisingly, these conformational sensors reveal a previously unappreciated level of diversity in GPCR-{beta}arr complexes that extends beyond the current framework of affinity-based classification and phosphorylation-code-based interaction patterns. More importantly, this conformational diversity arising from spatial signature of phosphorylation sites manifests directly in the form of distinct functional outcomes, including even opposite contribution of {beta}arrs in signal-transduction for different receptors. Taken together, these findings uncover that despite an overall similar interaction and trafficking patterns; critical structural and functional differences exist in {beta}arr complexes for different GPCRs that define and fine-tune receptor-specific downstream responses.

cell biology↗

bioRxiv2019

Partial ligand-receptor engagement yields functional bias at the human complement receptor, C5aR1

The human complement component, C5a, binds two different seven transmembrane receptors termed as C5aR1 and C5aR2. C5aR1 is a prototypical G protein-coupled receptor that couples to Gi sub-family of heterotrimeric G proteins and {beta}-arrestins ({beta}arr) following C5a stimulation. Peptide fragments derived from the carboxyl-terminus of C5a can still interact with the receptor, albeit with lower affinity, and can act as agonists or antagonists. However, whether such fragments might display ligand bias at C5aR1 remains unexplored. Here, we compare C5a and a modified C-terminal fragment of C5a, C5apep, in terms of G protein coupling, {beta}arr recruitment, endocytosis and ERK1/2 MAP kinase activation at the human C5aR1. We discover that C5apep acts as a full-agonist for G protein coupling, while only displaying partial agonism for {beta}arr recruitment. We also observe that whilst C5apep is significantly less efficient in inducing C5aR1 endocytosis compared to C5a, it exhibits robust activation of ERK1/2 phosphorylation at levels similar to C5a. Interestingly, C5apep displays full-agonist efficacy with respect to inhibiting LPS induced IL-6 secretion in human macrophages, but its ability to induce human neutrophil migration is substantially lower compared to C5a. Taken together, our findings reveal ligand-bias at C5aR1, not only with respect to transducer-coupling and receptor trafficking but also in terms of cellular responses. Our findings therefore establish a framework to explore additional levels of biased signaling and biased ligands at C5aR1 with therapeutic potential. More generally, our findings may be extended to discover biased ligands for the broad sub-family of chemokine GPCRs which also interact with chemokine ligands through a biphasic mechanism.

biochemistry↗