Biology subjects

Lambert, N. A.

Publications and source records attributed to Lambert, N. A..

7 recordsLinked to original sources

bioRxiv2026

Local GPCR density tips the balance of μ-opioid receptor trafficking

The extent to which local GPCR surface density governs engagement of downstream signaling and trafficking pathways remains unclear. Using single-particle tracking of the -opioid receptor (MOR), we show that receptor density differentially regulates G protein signaling and GRK2/3-{beta}-arrestin-dependent receptor trafficking. At low surface density, MORs activate G proteins but fail to enter clathrin-coated structures despite the presence of endogenous GRK2/3 and {beta}-arrestin. Increasing MOR density, co-expressing other class A GPCRs, or elevating GRK2 or {beta}-arrestin abundance rescues agonist-induced MOR trafficking. In contrast, the class B GPCR V2R blocks MOR trafficking at both low and high MOR densities. These results support a model in which increasing class A GPCR density, despite worsening effector-to-receptor stoichiometry, promotes trafficking by forming an affinity matrix that enables reversible GRK2/3 and {beta}-arrestin interactions to be productively used by neighboring receptors in a density-dependent manner, whereas class B GPCRs sequester {beta}-arrestin and block trafficking.

biophysics↗

bioRxiv2026

Gβγ engages PLCβ3 at multiple sites to reorient and facilitate its activation

Phospholipase C {beta} (PLC{beta}) enzymes are activated by heterotrimeric G protein subunits, increasing hydrolysis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. All four human PLC{beta} isoforms (PLC{beta}1-4) are activated by Gq, while PLC{beta}1-3 are activated to varying extents by G{beta}{gamma}. The binding sites for Gq on PLC{beta} are well-established and much has been learned about its mechanism of activation, but comparatively little is known about G{beta}{gamma}-dependent activation. In this work, we used cryo-electron microscopy (cryo-EM) single particle analysis (SPA), functional assays, and bioluminescence resonance energy transfer (BRET) to investigate how G{beta}{gamma} interacts with PLC{beta}3 in concert with activated Gq to regulate phospholipase activity. G{beta}{gamma} heterodimers bind multiple surfaces of PLC{beta}3 to promote activation but alone do not recruit the enzyme to the plasma membrane. Instead, G{beta}{gamma} facilitates activation by Gq, most likely by reorienting the phospholipase catalytic site at the membrane to maximize PI(4,5)P2 hydrolysis and downstream Ca2+ release. Cell-based functional assays demonstrate that G{beta}{gamma} is required for maximal PLC{beta}3 activation even when Gq heterotrimers are the sole source of G{beta}{gamma}. Together, these findings demonstrate that G{beta}{gamma} acts as a critical positive allosteric modulator that regularly acts in concert with Gq to activate PLC{beta}3 at the plasma membrane.

biochemistry↗

bioRxiv2024

Amylin receptor subunit interactions are modulated by agonists and determine signaling

Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMY1R, AMY2R, and AMY3R are heterodimers consisting of the G protein-coupled calcitonin receptor (CTR) paired with a RAMP1, -2, or -3 accessory subunit, respectively, which increases amylin potency. Little is known about AMYR subunit interactions and their role in signaling. Here, we show that the AMYRs have distinct basal subunit equilibriums that are modulated by peptide agonists and determine the cAMP signaling phenotype. Using a novel biochemical assay that resolves the AMYR heterodimers and free subunits, we found that the AMY1/2R subunit equilibriums favored free CTR and RAMP1/2, and rat amylin and CGRP agonists promoted subunit association. A stronger CTR-RAMP3 transmembrane domain interface yielded a more stable AMY3R, and human and salmon calcitonin agonists promoted AMY3R dissociation. Similar changes in subunit association-dissociation were observed in live cell membranes, and G protein coupling and cAMP signaling assays showed how these altered signaling. Our findings reveal regulation of heteromeric GPCR signaling through subunit interaction dynamics.

biochemistry↗

bioRxiv2024

Inefficient Endocytosis Limits Heterotrimeric G Protein Abundance on Endosomes

Classical G protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.

pharmacology and toxicology↗

bioRxiv2022

Nucleotide-decoupled G proteins reveal the role of G protein conformation in receptor-G protein selectivity

G protein-coupled receptors (GPCRs) selectively activate at least one of the four families of heterotrimeric G proteins to transduce environmental cues, but the mechanistic basis of coupling selectivity remains unclear. Structural studies have emphasized structural complementarity of GPCR complexes with nucleotide-free G proteins, but it has also been suggested that selectivity may be determined by intermediate activation processes that occur prior to nucleotide release. To test these ideas we have studied coupling to nucleotide- decoupled G protein variants, which can adopt conformations similar to receptor-bound G proteins without the need for nucleotide release. We find that selectivity is significantly degraded when nucleotide release is not required for GPCR-G protein complex formation, to the extent that most GPCRs interact with most nucleotide-decoupled G proteins. These findings demonstrate the absence of absolute structural incompatibility between most GPCRs and G proteins, and are consistent with the hypothesis that high-energy intermediate state complexes are involved in coupling selectivity.

pharmacology and toxicology↗

bioRxiv2021

Constitutive G protein coupling profiles of understudied orphan GPCRs

A large number of GPCRs are potentially valuable drug targets but remain understudied. Many of these lack well-validated activating ligands and are considered "orphan" receptors, and G protein coupling profiles have not been defined for many orphan GPCRs. Here we asked if constitutive receptor activity can be used to determine G protein coupling profiles of orphan GPCRs. We monitored nucleotide-sensitive interactions between 48 understudied orphan GPCRs and five G proteins (240 combinations) using bioluminescence resonance energy transfer (BRET). No receptor ligands were used, but GDP was used as a common G protein ligand to disrupt receptor-G protein complexes. Constitutive BRET between the same receptors and {beta}-arrestins was also measured. We found sufficient GDP-sensitive BRET to generate G protein coupling profiles for 22 of the 48 receptors we studied. Altogether we identified 48 coupled receptor-G protein pairs, many of which have not been described previously. We conclude that receptor-G protein complexes that form spontaneously in the absence of guanine nucleotides can be used to profile G protein coupling of constitutively active GPCRs. This approach may prove useful for studying G protein coupling of other GPCRs for which activating ligands are not available.

pharmacology and toxicology↗

bioRxiv2019

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric bias into the signaling system. However, the core underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of downstream readouts through which receptor-proximal interactions are typically inferred. Here we describe a scalable cell-based assay to overcome this challenge, based on the use of engineered GPCR-interacting proteins as orthogonal biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between protein probe recruitment produced by chemically distinct opioid ligands in living cells. We then show how the selective recruitment applies to GRK2, a biologically relevant opioid receptor regulator protein, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.

cell biology↗