Biology subjects

Picard, L.-P.

Publications and source records attributed to Picard, L.-P..

3 recordsLinked to original sources

bioRxiv2026

Local Confinement within Plasma Membrane Nanodomains Drives Constitutive Activity of GPCRs

Many G protein coupled receptors (GPCRs) exhibit constitutive (basal) activity, where they can signal in the absence of ligand binding through spontaneous conformational transitions that facilitate G protein coupling and downstream signaling. This intrinsic baseline activity is critical for cellular homeostasis and can be modulated by the receptors conformational ensemble, membrane organization, and interactions with intracellular effectors. In this study, we use live-cell signaling assays, fluorescence cross-correlation spectroscopy (FCCS), and single-particle tracking (SPT) to investigate how membrane organization influences the basal activity of two class A GPCRs: the M1 muscarinic receptor (M1R) and the adenosine A2A receptor (A2AR). In live-cell signalling assays, M1R showed minimal agonist-independent Ca{superscript 2} responses, while A2AR exhibited significant basal cAMP production that was eliminated by an inverse agonist. FCCS showed that, without ligand, only a small portion of M1R co-diffuses with its cognate G11 protein, whereas a much larger fraction of A2AR co-diffuses with the GS protein. SPT revealed that A2AR, but not M1R, is enriched in slowly diffusing, confined states with spatial scales around 150-200 nm and sensitivity to cholesterol- and raft-modulating agents, consistent with localization in lipid-raft nanodomains. Dual-color tracking and diffusion mapping demonstrated that a significant portion of A2AR and GS share confinement domains under basal conditions, while M1R and G11 only show such co-confinement in the active state. These findings support a model where the co-confinement of GPCRs and G proteins within plasma membrane nanodomains--rather than stable pre-coupled RG complexes-- determines the level of constitutive GPCR activity.

biophysics↗

bioRxiv2025

Isoform-Dependent Loss- and Gain-of-Function of the Gαs K53N Variant in Human Disease

The K53N mutation in Gs has been identified in patients with Albrights Hereditary Osteodystrophy (AHO), pseudohypoparathyroidism type 1A (PHP1a), and dilated cardiomyopathy; however, its molecular mechanism remains unclear. Here, we characterize the molecular, cellular, and physiological consequences of the K53N mutation in both long and short isoform of Gs. Biochemical analyses reveal that K53N disrupts nucleotide exchange and GTP hydrolysis, rendering both the short (Gs-S) and long (Gs-L) isoforms unresponsive to activation by G protein-coupled receptors (GPCRs) or cholera toxin. Both isoforms display a loss-of-function phenotype, failing to trigger cAMP production in response to {beta}2-adrenergic, parathyroid hormone, or vasopressin receptor stimulation. Notably, only the long isoform (Gs-L K53N) displays constitutive, receptor-independent cAMP generation. The mutation also reduces protein stability, weakens G{beta}{gamma} subunit interaction, and reduces plasma membrane localization. In neonatal rat ventricular cardiomyocytes, K53N impairs cAMP signaling and exerts dominant-negative effects on isoproterenol-induced responses. Strikingly, only Gs-L K53N abolishes isoproterenol-stimulated calcium release, directly implicating this isoform in the pathogenesis of cardiomyopathy. Collectively, these findings identify K53N as a unique Gs mutation that confers both loss- and gain-of-function properties in an isoform-specific manner, providing mechanistic insight into its complex pathogenicity in endocrine and cardiac tissues.

pharmacology and toxicology↗

bioRxiv2021

Allosteric modulation of the adenosine A2A receptor by cholesterol

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptors active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the observed cholesterol allostery in A2AR is likely a result of indirect membrane effects through cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

biophysics↗