Biology subjects

Blythe, E. E.

Publications and source records attributed to Blythe, E. E..

4 recordsLinked to original sources

bioRxiv2025

Conformational biosensors delineate endosomal G protein regulation by GPCRs

Many GPCRs trigger a second phase of G protein-coupled signaling from endosomes after initiating signaling from the plasma membrane. This inherently requires receptors to increase the concentration of active-state G proteins on the endosome membrane, but how this is achieved remains incompletely understood. We addressed this question by dissecting the regulation of G protein abundance and activity on endosomes following activation of several Gs-coupled GPCRs-the {beta}2-adrenergic receptor, the VIP-1 receptor, and the adenosine 2b receptor-that are natively co-expressed and differ in their ability to internalize after activation. We first verify GPCR-triggered redistribution of Gs from the plasma membrane to a mixed population of intracellular membranes, including endosomes, that is both reversible after receptor inactivation and triggered irrespective of the ability of the GPCR to internalize. We next show that GPCRs trigger this redistribution process at native expression levels and describe a method, using conformational biosensors, to detect endosomal activation of endogenous Gs. Applying this method, we show that GPCR-mediated production of active-state Gs on endosomes depends on receptor endocytosis, whereas increasing the net amount of Gs on endosomes does not. Our results support a model for Gs regulation on endosomes mediated by two spatially separated receptor coupling events-one at the plasma membrane controlling endosomal Gs abundance and another at endosomes controlling Gs activity. Additionally, our results reveal location-bias in the selectivity of G protein activation on endosomes that is differentially programmed by GPCRs in a receptor-specific manner.

cell biology↗

bioRxiv2025

Endocytosis sculpts distinct cAMP signal transduction by endogenously coexpressed GPCRs

Many G protein-coupled receptors (GPCRs) initiate a second phase of signaling after activation-induced endocytosis, but GPCRs vary considerably in their ability to internalize when activated. Here we show that this fundamental trafficking difference distinguishes the downstream signaling profiles of natively co-expressed GPCRs through the cAMP cascade. We focus on signaling to the nucleus stimulated by three different Gs-coupled GPCRs that are each endogenously co-expressed in human embryonic kidney cells but differ in their ability to internalize after activation: the adenosine-2B receptor that does not detectably internalize, the vasoactive intestinal peptide receptor-1 that internalizes very rapidly, and the {beta}2-adrenergic receptor that internalizes less rapidly. We show that each GPCR produces a distinct signaling profile differentiated by endocytosis. Our results support a model in which endocytosis compresses chemical information sensed by distinct GPCRs into a spatiotemporal cAMP code by setting receptor-specific differences in the amount and duration of cAMP production from endosomes relative to the plasma membrane and that this is decoded downstream in the pathway through sequential layers of processing by cytoplasmic and nuclear PKA activities. We propose that this biological information processing strategy has parallels to how computational encoder-decoder (autoencoder) networks denoise and recognize latent patterns in complex electrical signals.

cell biology↗

bioRxiv2022

A discrete mode of endosomal GPCR signaling that does not require β-arrestin

Many GPCRs are now recognized to initiate a second phase of G protein (Gs) -dependent signaling through the cAMP cascade after endocytosis. A prevailing current view is that endocytosis-promoted signaling from GPCRs is inherently {beta}-arrestin-dependent because {beta}-arrestin is necessary for receptors to internalize and, for some GPCRs, it also promotes Gs activation on endosomes. Here we revise this view by showing that the vasoactive intestinal peptide receptor 1 (VIPR1), a prototypic secretin-family polypeptide hormone receptor, remains bound to {beta}-arrestin after endocytosis but does not require {beta}-arrestin either to internalize or to generate an endosomal signal. {beta}-arrestin instead resolves the endosomal signal into a temporally separated cAMP peak, and it does so by attenuating signaling from the plasma membrane without detectably affecting the endosomal response. The mechanistic basis for this location-specific difference in {beta}-arrestin function is the formation of distinct VIPR1/{beta}-arrestin complexes at each location. The signal-attenuating complex formed at the plasma membrane does not require GRK-mediated phosphorylation of receptors, while the signaling-neutral complex present on the endosome membrane, in contrast, is GRK-dependent. To our knowledge, the present results provide the first direct demonstration that endosomal GPCR signaling can occur in the complete absence of {beta}-arrestin. They also reveal a discrete role of {beta}-arrestin in sculpting the spatiotemporal profile of cellular GPCR - G protein signaling through the location-specific formation or remodeling of GPCR/{beta}-arrestin complexes.

cell biology↗

bioRxiv2022

Profiling the diversity of agonist-selective effects on the proximal proteome environment of G protein-coupled receptors

The mu opioid receptor (OR), a prototypic member of the large G protein-coupled receptor (GPCR) family, represents an important target of therapeutic and abused drugs. To date, most of our understanding of OR activity has focused on signal transducers and regulatory molecules including G proteins, GPCR kinases, and beta-arrestins. Yet it is clear that signaling through the OR is coordinated by additional proteins recruited into the proximal interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here, we implement a quantitative proteomics pipeline leveraging the chemical diversity of OR agonists and APEX-based proximity labeling to investigate the protein networks that underlie OR signaling. We leverage a novel computational framework to extract subcellular location, trafficking, and functional partners of GPCR activity from the proximity labeling datasets. Applying this unbiased, systematic approach to the OR, we demonstrate that opioid agonists exert differences in the OR proximal proteome mediated by endocytosis and subsequent endosomal sorting, exemplified by VPS35 and COMMD3. Moreover, we identify two novel OR network components, EYA4 and KCTD12, that are recruited into the receptor proximal network irrespective of the activating ligand and independent of receptor trafficking but based on receptor-triggered G protein activation. We provide functional evidence that these network components form a previously unrecognized buffering system for G protein activity which broadly modulates cellular GPCR signaling.

systems biology↗