Biology subjects

Yen, Y.-C.

Publications and source records attributed to Yen, Y.-C..

3 recordsLinked to original sources

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↗