Constitutive internalization across therapeutically targeted GPCRs correlates with constitutive activity

Abstract

While the physiological function and mechanisms of agonist-dependent G proteincoupled receptor (GPCR) internalization have been extensively studied, the functional characterization of constitutive internalization of these critically important receptors has received less attention. Here we relate the constitutive internalization of more than 30 therapeutically targeted GPCRs to their agonist-induced internalization. The constitutive internalization ranges from levels of bulk membrane endocytosis in some cases to levels of agonist-induced internalization for other receptors.Moreover, for receptors with high constitutive internalization this occludes further agonist-induced internalization. Additionally, Gq-coupled GPCRs show a significantly higher rate of constitutive internalization than Gs-and Gi-coupled receptors.Finally, we consolidate the proposed link between the constitutive internalization, as assessed by a cytometry-based assay, and the constitutive activity of these receptors, as previously reported by a β-arrestin recruitment assay across the range of pharmacologically relevant receptors. In summary, we provide a quantitative comparison of GPCR internalization across a range of pharmacologically relevant receptors providing generalized insight into the relations between constitutive internalization, constitutive activity and agonist-induced internalization, which has so far relied on mutational studies in individual receptors. K E Y W O R D Sclathrin-independent endocytosis, constitutive endocytosis, endosomal signalling, PRESTO-Tango, β-arrestin signalling

Constitutive internalisation of EP2 differentially regulates G protein signalling

“…The present study confirms previous reports that EP2 does not undergo agonist-driven internalisation ( Desai et al 2000 , Penn et al 2001 ) but identifies constitutive internalisation of EP2. This is supported by previous studies which find that generally, there is an inverse relationship between constitutive and agonist-induced internalisation ( Hendrik Schmidt et al 2020 ). This has been overlooked for EP2, as studies have focused exclusively on agonist-driven internalisation ( Desai et al 2000 , Penn et al 2001 ).…”

UR-DEBa242: A Py-5-Labeled Fluorescent Multipurpose Probe for Investigations on the Histamine H3 and H4 Receptors

“…Unexpectedly, 26 , an inverse agonist at the hH 4 R in a β-arrestin recruitment assay (Table ), was internalized in a receptor-dependent manner. This may be taken as a hint to constitutive endocytosis of the hH 4 R by β-arrestin- or even clathrin-independent mechanisms, a process already described for various GPCRs (e.g., M 3 muscarinic receptor, β 2 adrenergic receptor, or 5-HT 2A serotonin receptor , ). Furthermore, the observed internalization of the fluorescent probe 26 was in agreement with the comparatively high nonspecific binding of 26 in flow cytometric saturation binding experiments (Figure S16).…”

Membrane curvature association of amphipathic helix 8 drives constitutive endocytosis of GPCRs

“…e Heatmaps of all individual GPCR C-terminal liposome binding profiles within the six clusters and profile plots of the median fluorescence intensity (line above) (see S19 from detailed view). f Linear correlation (R² = 0.83, p <0.0001) between MIP of H8 from clusters 2-5, based on sequences (Figure S20) in most pharmacologically relevant GPCRs and their respective endocytic rate (relative to Tac) determined by flow cytometry 35 . Mean ± SEM, N=3.…”

“…The experimentally derived membrane binding profiles enabled us to calculate MIP values for the regions corresponding to bona fide H8s (clusters 2-5, Figure S20), while the lack of membrane binding in cluster 1, precluded calculation of MIP values. We previously characterized the constitutive internalization of a range of pharmacologically relevant GPCRs 35 and here asked if this internalization could be explained by MIP values of the regions defined by their membrane binding profiles. Constitutive internalization significantly correlated with MIP (cluster 2-5) (Figure 4F), however, traditional biophysical measures, alternative to MIP, failed to explain constitutive internalization (Figure S21A-C).…”