Biology subjects

Devost, D.

Publications and source records attributed to Devost, D..

3 recordsLinked to original sources

bioRxiv2023

Structure and dynamics of a pentameric KCTD5/Cullin3/Gβγ E3 ubiquitin ligase complex

Heterotrimeric G proteins can be regulated by post-translational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain (CTD), engages CUL3 to form the central scaffold of a cullin- RING E3 ligase complex (CRL3KCTD5) that ubiquitylates G{beta}{gamma} and reduces G{beta}{gamma} protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/G{beta}1{gamma}2 assembly reveals a highly dynamic complex with rotations of over 60{degrees} between the KCTD5BTB/CUL3NTD and KCTD5CTD/G{beta}{gamma} moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate G{beta}{gamma} in an E3-E3 super-assembly, and extension of the structure to include full- length CUL3 with RBX1 and an ARIH1[~]ubiquitin conjugate reveals that some conformational states position the ARIH1[~]ubiquitin thioester bond to within 10 [A] of lysine-23 of G{beta} and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/G{beta}{gamma} complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex. Significance StatementIn humans, [~]600 enzyme complexes can carry out protein ubiquitylation, and the most abundant class of these are the cullin3-RING-ligase complexes (CRL3s). CRL3s are multiprotein complexes built around a BTB/cullin3 core, and the incorporation of different BTB proteins into this scaffold results in distinct architectures that ubiquitylate a wide range of substrates. In most cases, it is not known how the complexes are tuned to their substrates. We show that the BTB protein KCTD5 is the central organizer in a CRL3KCTD5 complex, and that the architecture and internal dynamics of KCTD5 are essential for positioning a G{beta}{gamma} substrate protein near an activated ubiquitin for the transfer reaction. This explains how KCTD5 targets G{beta}{gamma} for proteasomal degradation and regulates cellular activities.

biochemistry↗

bioRxiv2022

Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of {beta}-arrestin ({beta}arr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for {beta}arrs, agonist-stimulation first drives the translocation of {beta}arrs 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 {beta}arr translocation to endosomes although {beta}arrs 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 {beta}arr1 conformation, rescues endosomal translocation of {beta}arr1 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 {beta}arr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of {beta}arr1 with {beta}2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of {beta}arr1. 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. SignificanceThe interaction of G protein-coupled receptors (GPCRs) with {beta}-arrestins ({beta}arrs) is a critical step in their regulatory and signaling paradigms. While intrabodies that bind to GPCRs, G proteins and {beta}arrs 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 {beta}arr1 can allosterically enhance endosomal trafficking of {beta}arr1 and agonist-induced ERK1/2 MAP kinase activation. This intrabody promotes an active-like {beta}arr1 conformation and enhances the interaction of {beta}2-adaptin with {beta}arr1. Our findings establish a conceptual framework to allosterically modulate protein-protein interactions in GPCR signaling cascade to modulate their trafficking and signaling responses.

biochemistry↗

bioRxiv2021

Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization. This might be an evolutionary adaptation that enables LH surge generation and ovulation. To test this idea, we fused the chicken GnRHR Ctail to the endogenous murine GnRHR in a transgenic model. The LH surge was blunted, but not blocked in these mice. In contrast, they showed reductions in FSH production, ovarian follicle development, and fertility. Addition of the Ctail altered the nature of agonist-induced calcium signaling required for normal FSH production. The loss of the GnRHR Ctail during mammalian evolution is unlikely to have conferred a selective advantage by enabling the LH surge. The adaptive significance of this specialization remains to be determined.

physiology↗