A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis

Ghosh, Eshan; Srivastava, Ashish; Baidya, Mithu; Kumari, Punita; Dwivedi, Hemlata; Nidhi, Kumari; Ranjan, Ravi; Dogra, Shalini; Koide, Shohei; Yadav, Prem N.; Sidhu, Sachdev S.; Shukla, Arun K.

doi:10.1038/nnano.2017.188

Cited by 42 publications

(49 citation statements)

References 58 publications

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“…Leading up to this point, our data based on Fab30 recognition suggest potential conformational difference between receptor-bound βarr1 and 2. In order to corroborate these findings further, we tested a series of additional Fabs that we have recently generated and characterized to selectively recognize V 2 Rpp-bound βarr1 (Ghosh et al, 2017). Similar to Fab30, these additional Fabs also interacted comparably with V 2 Rpp-bound βarr1 and 2 (Figure S8D), and efficiently recognized the complex of βarr1 with activated and phosphorylated receptor (Figure S8E).…”

Section: Resultsmentioning

confidence: 94%

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

Ghosh

Dwivedi

Baidya

et al. 2019

Preprint

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SummaryDesensitization, signaling and trafficking of G protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, β-arrestins (βarrs). The two isoforms of βarrs (βarr1 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 βarr isoforms is still lacking. Using a set of complementary approaches including antibody fragment based conformational sensors, we discover structural differences between βarr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain swapped chimeras of βarrs display robust complementation in functional assays thereby, linking the structural differences between the receptor-bound βarr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of βarr isoforms to drive distinct functional outcomes, and underscore the importance of integrating this aspect in the current framework of biased agonism.

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Section: Resultsmentioning

confidence: 94%

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

Ghosh

Dwivedi

Baidya

et al. 2019

Preprint

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“…A phosphopeptide corresponding to the carboxylterminus of the human vasopressin V2 receptor, referred to as V2Rpp, has been used extensively as a surrogate to induce active βarr conformation in-vitro (22)(23)(24)(25). We have previously generated and characterized a set of synthetic antibody fragments (FABs) that selectively recognize V2Rpp-bound βarr1 (26).…”

Section: Synthetic Antibody Fragments Report the Formation Of β2v2r-βmentioning

confidence: 99%

Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G-protein–coupled receptors

Baidya

Kumari

Dwivedi-Agnihotri

et al. 2020

Journal of Biological Chemistry

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Agonist stimulation of G protein–coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR–βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR–βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR–βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR–βarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments (ScFvs) and used them to monitor the localization and trafficking of βarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR–βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

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“…Next to receptor-specific intrabodies, more generic intrabodies can target GPCR-interaction partners. For example, intrabodies targeting b-arrestin can act as generic GPCR endocytosis inhibitors without affecting b-arrestin-mediated signaling (Ghosh et al, 2017). Another study generated an intrabody that targeted G bg and competed with the binding G bg -interaction partners while having no effect on G aq -or G as -mediated signaling (Gulati et al, 2018).…”

Section: Single-domain Antibodies As Therapeutics and Tools In Gpcr Rmentioning

confidence: 99%

Antibodies Targeting Chemokine Receptors CXCR4 and ACKR3

et al. 2019

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Dysregulation of the chemokine system is implicated in a number of autoimmune and inflammatory diseases, as well as cancer. Modulation of chemokine receptor function is a very promising approach for therapeutic intervention. Despite interest from academic groups and pharmaceutical companies, there are currently few approved medicines targeting chemokine receptors. Monoclonal antibodies (mAbs) and antibody-based molecules have been successfully applied in the clinical therapy of cancer and represent a potential new class of therapeutics targeting chemokine receptors belonging to the class of G protein-coupled receptors (GPCRs). Besides conventional mAbs, single-domain antibodies and antibody scaffolds are also gaining attention as promising therapeutics. In this review, we provide an extensive overview of mAbs, singledomain antibodies, and other antibody fragments targeting CXCR4 and ACKR3, formerly referred to as CXCR7. We discuss their unique properties and advantages over smallmolecule compounds, and also refer to the molecules in preclinical and clinical development. We focus on singledomain antibodies and scaffolds and their utilization in GPCR research. Additionally, structural analysis of antibody binding to CXCR4 is discussed. SIGNIFICANCE STATEMENT Modulating the function of GPCRs, and particularly chemokine receptors, draws high interest. A comprehensive review is provided for monoclonal antibodies, antibody fragments, and variants directed at CXCR4 and ACKR3. Their advantageous functional properties, versatile applications as research tools, and use in the clinic are discussed.

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A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis

Cited by 42 publications

References 58 publications

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

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

Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G-protein–coupled receptors

Antibodies Targeting Chemokine Receptors CXCR4 and ACKR3

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