Rasmus Herlo

Cellular signaling relies on the activity of transmembrane receptors and their presentation on the cellular surface. Their continuous insertion in the plasma membrane is balanced by constitutive and activity-dependent internalization, which is orchestrated by adaptor proteins recognizing semispecific motifs within the receptors’ intracellular regions. Here, we describe a complementary trafficking mechanism for G protein–coupled receptors (GPCRs) that is evolutionary conserved and refined. This mechanism relies on the insertion of their amphipathic helix 8 into the inner leaflet of lipid membranes, orthogonal to the transmembrane helices. These amphipathic helices dictate subcellular localization of the receptors and autonomously drive their endocytosis by cooperative assembly and association with areas of high membrane curvature. The strength of helix 8 membrane insertion propensity quantitatively predicts the rate of constitutive internalization of GPCRs. This discovery advances our understanding of membrane protein trafficking and highlights a principle of receptor-lipid interactions that may have broad implications for cellular signaling and therapeutic targeting.

Local neural activation drives regional increase of cerebral blood flow (CBF), in a phenomenon known as functional hyperemia. Astrocytes, which enwrap cerebral blood vessels and respond to neuronal activity through their G protein–coupled receptors (GPCRs), play a vital role in brain energy metabolism. Although astrocytic calcium (Ca ²⁺ ) signaling has been widely studied in relation to neurovascular coupling, the role of cyclic adenosine monophosphate (cAMP), another key second messenger of GPCRs, on CBF has not been established. In this study, we explored the effects of optogenetically induced astrocytic cAMP elevation on CBF. We engineered adeno-associated viral vectors (AAVs) to express a bacterial photoactivated adenylyl cyclase in astrocytes, which triggers an increase in cAMP upon blue light stimulation. Opto-stimulation also elevated astrocytic Ca ²⁺ , albeit with a delayed onset under mild stimulation. In vivo imaging of anesthetized and awake wild-type mice through a thinned skull preparation revealed that optogenetically induced astrocytic cAMP elevation led to pronounced arteriole dilation, with a latency of 1.8 s and maximal dilation reached within 10 s in the awake state and slower response under anesthesia. Mild opto-stimulation causing sensory-level cAMP elevations was sufficient to induce arteriole dilation. This effect was preserved in IP ₃ receptor type 2-knockout (IP ₃ R2 ^−/− ) mice, indicating a mechanism independent of GPCR-induced intracellular Ca ²⁺ elevations. These findings highlight astrocytic cAMP as a key modulator of cerebral vasodilation, contributing to our understanding of local CBF regulation. This study opens broad avenues for understanding astrocyte-mediated control of CBF and its implications in neurological diseases characterized by dysregulated blood flow.

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.

Summary: Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that shape negatively charged curved lipid membranes during membrane remodeling processes. The BAR domain proteins ICA69, PICK1 and arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans -Golgi network. Here, we identify four coding variants in the PICK1 gene from a Danish whole-exome screening of diabetic patients, that all involve change of positively charged residues in the PICK1 BAR domain. All four coding variants failed to rescue the insulin content in INS-1E cells upon KD of endogenous PICK1. Moreover, two variants showed dominant negative properties. Interestingly, in vitro assays addressing the BAR domain function suggest that the coding variants compromised membrane binding capacity but increased capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin from ISGs during the maturation process and that the coding variants may cause premature budding possibly explaining their dominant negative function.

While the physiological function and mechanisms of agonist‐dependent G protein‐coupled 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.

The shape and composition of a membrane directly regulate the localization, activity, and signaling properties of membrane associated proteins. Proteins that both sense and generate membrane curvature, e.g., through amphiphilic insertion motifs, potentially engage in recursive binding dynamics, where the recruitment of the protein itself changes the properties of the membrane substrate. Simple geometric models of membrane curvature interactions already provide prediction tools for experimental observations, however these models are treating curvature sensing and generation as separated phenomena. Here, we outline a model that applies both geometric and basic thermodynamic considerations. This model allows us to predict the consequences of recursive properties in such interaction schemes and thereby integrate the membrane as a dynamic substrate. We use this combined model to hypothesize the origin and properties of tubular carrier systems observed in cells. Furthermore, we pinpoint the coupling to a membrane reservoir as a factor that influences the membrane curvature sensing and generation properties of local curvatures in the cell in line with classic determinants such as lipid composition and membrane geometry.