Biology subjects

Tesmer, J. J. G.

Publications and source records attributed to Tesmer, J. J. G..

9 recordsLinked to original sources

bioRxiv2023

Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4

PIP3-dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP3 and the heterotrimeric G{beta}{gamma} subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP3, we discovered that Ins(1,3,4,5)P4 (IP4) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1{middle dot}IP4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH-DEP1 and PH-4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP3-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

biochemistry↗

bioRxiv2023

ACKR3-arrestin2/3 complexes reveal molecular consequences of GRK-dependent barcoding

Atypical chemokine receptor 3 (ACKR3, also known as CXCR7) is a scavenger receptor that regulates extracellular levels of the chemokine CXCL12 to maintain responsiveness of its partner, the G protein-coupled receptor (GPCR), CXCR4. ACKR3 is notable because it does not couple to G proteins and instead is completely biased towards arrestins. Our previous studies revealed that GRK2 and GRK5 install distinct distributions of phosphates (or "barcodes") on the ACKR3 carboxy terminal tail, but how these unique barcodes drive different cellular outcomes is not understood. It is also not known if arrestin2 (Arr2) and 3 (Arr3) bind to these barcodes in distinct ways. Here we report cryo-electron microscopy structures of Arr2 and Arr3 in complex with ACKR3 phosphorylated by either GRK2 or GRK5. Unexpectedly, the finger loops of Arr2 and 3 directly insert into the detergent/membrane instead of the transmembrane core of ACKR3, in contrast to previously reported "core" GPCR-arrestin complexes. The distance between the phosphorylation barcode and the receptor transmembrane core regulates the interaction mode of arrestin, alternating between a tighter complex for GRK5 sites and heterogenous primarily "tail only" complexes for GRK2 sites. Arr2 and 3 bind at different angles relative to the core of ACKR3, likely due to differences in membrane/micelle anchoring at their C-edge loops. Our structural investigations were facilitated by Fab7, a novel Fab that binds both Arr2 and 3 in their activated states irrespective of receptor or phosphorylation status, rendering it a potentially useful tool to aid structure determination of any native GPCR-arrestin complex. The structures provide unprecedented insight into how different phosphorylation barcodes and arrestin isoforms can globally affect the configuration of receptor-arrestin complexes. These differences may promote unique downstream intracellular interactions and cellular responses. Our structures also suggest that the 100% bias of ACKR3 for arrestins is driven by the ability of arrestins, but not G proteins, to bind GRK-phosphorylated ACKR3 even when excluded from the receptor cytoplasmic binding pocket.

biochemistry↗

bioRxiv2023

A Ciliary SMOOTHENED-GRK2-PKA Signaling Pathway Initiates Hedgehog Signal Transduction

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the vertebrate primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

cell biology↗

bioRxiv2023

Molecular basis for Gβγ-mediated activation ofphosphoinositide 3-kinase γ

The conversion of PIP2 to PIP3 by phosphoinositide 3-kinase {gamma} (PI3K{gamma}) is a critical step in neutrophil chemotaxis and is essential for metastasis in many types of cancer. PI3K{gamma} is activated via directed interaction with G{beta}{gamma} heterodimers released from cell-surface G protein-coupled receptors (GPCRs) responding to extracellular signals. To resolve how G{beta}{gamma} activates PI3K{gamma}, we determined cryo-EM reconstructions of PI3K{gamma}-G{beta}{gamma} complexes in the presence of various substrates/analogs, revealing two distinct G{beta}{gamma} binding sites, one on the p110{gamma} helical domain and one on the C-terminal domain of the p101 subunit. Comparison of these complexes with structures of PI3K{gamma} alone demonstrates conformational changes in the kinase domain upon G{beta}{gamma} binding similar to those induced by Ras{middle dot}GTP. Assays of variants perturbing the two G{beta}{gamma} binding sites and interdomain contacts that change upon G{beta}{gamma} binding suggest that G{beta}{gamma} not only recruits the enzyme to membranes but also allosterically controls activity via both sites. Studies in a zebrafish model examining neutrophil migration are consistent with these results. These findings set the stage for future detailed investigation of G{beta}{gamma}-mediated activation mechanisms in this enzyme family and will aid in developing drugs selective for PI3K{gamma}.

biophysics↗

bioRxiv2023

Isoform Specific Regulation of Adenylyl Cyclase 5 by Gβγ

The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein Gs, but their response to G{beta}{gamma} regulation is isoform-specific. For example, AC5 is conditionally activated by G{beta}{gamma}. Here, we report cryo-EM structures of ligand-free AC5 in complex with G{beta}{gamma} and of a dimeric form of AC5 that could be involved in its regulation. G{beta}{gamma} binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the G{beta}{gamma} interaction with both purified proteins and cell-based assays. The interface with G{beta}{gamma} involves AC5 residues that are subject to gain-of-function mutations in humans with familial dyskinesia, indicating that the observed interaction is important for motor function. A molecular mechanism wherein G{beta}{gamma} either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core, is proposed. Because our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

biophysics↗

bioRxiv2023

Atypical Chemokine Receptor 3 'Senses' CXC Chemokine Receptor 4 Activation Through GPCR Kinase Phosphorylation

Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging action mediates the availability of the chemokine CXCL12 for the G protein-coupled receptor (GPCR) CXCR4 and requires phosphorylation of the ACKR3 C-terminus by GPCR kinases (GRKs). ACKR3 is phosphorylated by GRK2 and GRK5, but the mechanisms by which these kinases regulate the receptor are unresolved. Here we mapped the phosphorylation patterns and determined that GRK5 phosphorylation of ACKR3 dominates {beta}-arrestin recruitment and chemokine scavenging over GRK2. Co-activation of CXCR4 significantly enhanced phosphorylation by GRK2 through the liberation of G{beta}{gamma}. These results suggest that ACKR3 senses CXCR4 activation through a GRK2-dependent crosstalk mechanism. Surprisingly, we also found that despite the requirement for phosphorylation, and the fact that most ligands promote {beta}-arrestin recruitment, {beta}-arrestins are dispensable for ACKR3 internalization and scavenging, suggesting a yet to be determined function for these adapter proteins.

biochemistry↗

bioRxiv2020

Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

Insulin impairs {beta}2-adrenergic receptor ({beta}2AR) function through G protein-coupled receptor kinase 2 (GRK2) by phosphorylation but less is known about dephosphorylation mechanisms mediated by protein phosphatase 2A (PP2A). Pharmacologic or genetic inhibition of phosphoinositide 3-kinase {gamma} (PI3K{gamma}) unexpectedly resulted in significant reduction of insulin-mediated {beta}2AR phosphorylation. Interestingly, {beta}2AR-associated phosphatase activity was inhibited by insulin but was reversed by knock-down of PI3K{gamma} showing negative regulation of PP2A by PI3K{gamma}. Co-immunoprecipitation and surface plasmon resonance studies using purified proteins showed that GRK2 and PI3K{gamma} form a complex and could be recruited to {beta}2ARs as GRK2 interacts with insulin receptor substrate following insulin treatment. Consistently, {beta}-blocker pretreatment did not reduce insulin-mediated {beta}2AR phosphorylation indicating agonist- and G{beta}{gamma}-independent non-canonical regulation of receptor function. Mechanistically, PI3K{gamma} inhibits PP2A activity at the {beta}AR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A). Knock-down or CRISPR ablation of endogenous I2PP2A unlocked PP2A inhibition mediating {beta}2AR dephosphorylation showing an unappreciated acute regulation of PP2A in mediating insulin-{beta}2AR cross-talk. SummaryInsulin impairs {beta}2-adrenergic receptor ({beta}2AR) function through G protein-coupled receptor kinase 2 (GRK2). We show that insulin simultaneously inhibits protein phosphatase 2A (PP2A) sustaining {beta}2AR functional impairment. Unexpectedly, releasing PP2A inhibition by PI3K{gamma} preserves {beta}2AR function despite intact insulin-driven GRK2-mechanisms.

biochemistry↗

bioRxiv2019

High-throughput cryo-EM enabled by user-free preprocessing routines

The growth of single-particle cryo-EM into a mainstream structural biology tool has allowed for many important biological discoveries. Continued developments in data collection strategies alongside new sample preparation devices heralds a future where users will collect multiple datasets per microscope session. To make cryo-EM data processing more automatic and user-friendly, we have developed an automatic pipeline for cryo-EM data preprocessing and assessment using a combination of deep learning and image analysis tools. We have verified the performance of this pipeline on a number of datasets and extended its scope to include sample screening by the user-free assessment of the qualities of a series of datasets under different conditions. We propose that our workflow provides a decision-free solution for cryo-EM, making data preprocessing more generalized and robust in the high-throughput era as well as more convenient for users from a range of backgrounds.

biophysics↗

bioRxiv2018

Molecular basis for activation of lecithin:cholesterol acyltransferase by a compound that increases HDL cholesterol

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating small molecules are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of LCAT bound to a potent activator and an acyl intermediate-like inhibitor, thereby revealing an active conformation of LCAT and that the activator is bound exclusively to its membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and likely facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. In addition, our data better define the acyl binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients.

biochemistry↗