Agonist-induced Internalization Research Articles

β-arrestin1/2 are essential for embryonic lymphatic vessel development

Lymphatic dysfunction can lead to fetal loss due to interstitial edema known as hydrops fetalis during development and lymphedema in adulthood. Lymphedema affects an estimated 250 million individuals worldwide, yet there is no effective treatment available. Numerous studies have established G protein-coupled receptors (GPCRs) as key lymphangiogenic signaling pathways and ideal druggable therapeutical targets. β-arrestins (β-arrestin 1 and 2) are ubiquitously expressed cytosolic adaptor proteins which perform a wide range of functions in GPCR pathways, such as promoting agonist-induced internalization and forming signaling scaffolds with internalized GPCRs in endosomes. However, the roles of β-arrestins in lymphatic vessel development and function remain undefined. We hypothesize that β-arrestin1/2 are essential in embryonic lymphatic vessel development. To test this hypothesis, I used an inducible β-arrestin1fl/fl (Arrb1 fl/fl), β-arrestin2fl/fl (Arrb2 fl/fl);Prox1 CreERT2 mouse line. β-arrestin1 and 2 were deleted by oral gavage of tamoxifen at embryonic day 10.5 (E10.5) and E12.5, critical points for developmental lymphangiogenesis. Compared to Arrb1/2fl/flembryos, the Arrb1/2fl/fl;Prox1CreERT2 embryos exhibit profound hydrops fetalis, subcutaneous hemorrhages, and increased embryonic lethality between E14.5 and E16.5. At E15.5, the Arrb1/2fl/fl;Prox1CreERT2 embryos with edema show decreased weight indicating growth restriction compared to controls. Jugular lymphatic sacs are enlarged and filled with blood in Arrb1/2fl/fl;Prox1CreERT2 embryos compared to controls. Dermal lymphatic vessel length in the Arrb1/2fl/fl;Prox1CreERT2 embryos is shorter than controls. Collectively, our results demonstrate that loss of β-arrestin1/2 expression in lymphatics causes mid-gestational arrest in lymphatic development and fetal growth restriction, indicating that β-arrestins are required for developmental lymphangiogenesis in mammals. American Physiological Society postdoctoral Fellowship (to YT) and NIH R01 HL129086 (to KMC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Segregation of nascent GPCRs in the ER-to-Golgi transport by CCHCR1 via direct interaction.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins that share a common structural topology. When compared with agonist-induced internalization, how GPCRs are sorted and delivered to functional destinations after synthesis in the endoplasmic reticulum (ER) is much less well understood. Here, we demonstrate that depletion of coiled-coil α-helical rod protein 1 (CCHCR1) by siRNA and CRISPR-Cas9 significantly inhibits surface expression and signaling of α2A-adrenergic receptor (α2A-AR; also known as ADRA2A), without affecting α2B-AR. Further studies show that CCHCR1 depletion specifically impedes α2A-AR export from the ER to the Golgi, but not from the Golgi to the surface. We also demonstrate that CCHCR1 selectively interacts with α2A-AR. The interaction is mediated through multiple domains of both proteins and is ionic in nature. Moreover, mutating CCHCR1-binding motifs significantly attenuates ER-to-Golgi export, surface expression and signaling of α2A-AR. Collectively, these data reveal a novel function for CCHCR1 in intracellular protein trafficking, indicate that closely related GPCRs can be sorted into distinct ER-to-Golgi transport routes by CCHCR1 via direct interaction, and provide important insights into segregation and anterograde delivery of nascent GPCR members.

Insight into the mechanism of action of ORG27569 at the cannabinoid type one receptor utilising a unified mathematical model

Allosteric modulation of CB1 is therapeutically advantageous compared to orthosteric activation as it potentially offers reduced on-target adverse effects. ORG27569 is an allosteric modulator that increases orthosteric agonist binding to CB1 but decreases functional signalling. ORG27569 is characterised by a delay in disinhibition of agonist-induced cAMP inhibition (lag); however, the mechanism behind this kinetic lag is yet to be identified. We aimed to utilise a mathematical model to predict data and design in vitro experiments to elucidate mechanisms behind the unique signalling profile of ORG27569. The established kinetic ternary complex model includes the existence of a transitional state of CB1 bound to ORG27569 and CP55940 and was used to simulate kinetic cAMP data using NONMEM 7.4 and Matlab R2020b. These data were compared with empirical cAMP BRET data in HEK293 cells stably expressing hCB1. The pharmacometric model suggested that the kinetic lag in cAMP disinhibition by ORG27569 is caused by signal amplification in the cAMP assay and can be reduced by decreasing receptor number. This was confirmed experimentally, as reducing receptor number through agonist-induced internalisation resulted in a decreased kinetic lag by ORG27569. ORG27569 was found to have a similar interaction with CP55940 and the high efficacy agonist WIN55,212-2, and was suggested to have lower affinity for CB1 bound by the partial agonist THC compared to CP55940. Allosteric modulators have unique signalling profiles that are often difficult to interrogate exclusively in vitro. We have used a combined mathematical and in vitro approach to prove that ORG27569 causes a delay in disinhibition of agonist-induced cAMP inhibition due to large receptor reserve in this pathway. We also used the pharmacometric model to investigate the common phenomenon of probe dependence, to propose that ORG27569 binds with higher affinity to CB1 bound by high efficacy orthosteric agonists.

The GPCR adaptor protein Norbin regulates S1PR1 trafficking and the morphology, cell cycle and survival of PC12 cells

Norbin is an adaptor protein that binds numerous G protein-coupled receptors (GPCRs), is highly expressed in neurons, and is essential for a functioning nervous system in rodent models. Yet, beyond its control of neurite outgrowth and synaptic plasticity, few cellular roles of Norbin have been investigated to date. Furthermore, while Norbin is known to regulate the steady-state cell surface levels of several GPCRs, only in one case has the protein been shown to control the agonist-induced receptor internalisation which serves to attenuate GPCR signalling. Here, we generated a Norbin-deficient PC12 cell line which enabled us to study both the cellular functions of Norbin and its roles in GPCR trafficking and signalling. We show that Norbin limits cell size and spreading, and is required for the growth, viability and cell cycle progression of PC12 cells. We also found that Norbin regulates both the steady-state surface level and agonist-induced internalisation of the GPCR sphingosine-1-phosphate receptor 1 (S1PR1) in these cells, suggesting that its role in agonist-dependent GPCR trafficking is more widespread than previously appreciated. Finally, we show that Norbin limits the S1P-stimulated activation of Akt and p38 Mapk, and is required for the activation of Erk in PC12 cells. Together, our findings provide a better understanding of the cellular functions of Norbin and its control of GPCR trafficking.

Amphetamine pretreatment blunts dopamine-induced D2/D3-receptor occupancy by an arrestin-mediated mechanism: A PET study in internalization compromised mice

While all reversible receptor-targeting radioligands for positron emission tomography (PET) can be displaced by competition with an antagonist at the receptor, many radiotracers show limited occupancies using agonists even at high doses. [11C]Raclopride, a D2/D3 receptor radiotracer with rapid kinetics, can identify the direction of changes in the neurotransmitter dopamine, but quantitative interpretation of the relationship between dopamine levels and radiotracer binding has proven elusive. Agonist-induced receptor desensitization and internalization, a homeostatic mechanism to downregulate neurotransmitter-mediated function, can shift radioligand-receptor binding affinity and confound PET interpretations of receptor occupancy. In this study, we compared occupancies induced by amphetamine (AMP) in drug-naive wild-type (WT) and internalization-compromised β-arrestin-2 knockout (KO) mice using a within-scan drug infusion to modulate the kinetics of [11C]raclopride. We additionally performed studies at 3 h following AMP pretreatment, with the hypothesis that receptor internalization should markedly attenuate occupancy on the second challenge, because dopamine cannot access internalized receptors. Without prior AMP treatment, WT mice exhibited somewhat larger binding potential than KO mice but similar AMP-induced occupancy. At 3 h after AMP treatment, WT mice exhibited binding potentials that were 15 % lower than KO mice. At this time point, occupancy was preserved in KO mice but suppressed by 60 % in WT animals, consistent with a model in which most receptors contributing to binding potential in WT animals were not functional. These results demonstrate that arrestin-mediated receptor desensitization and internalization produce large effects in PET [11C]raclopride occupancy studies using agonist challenges.

G protein–receptor kinases 5/6 are the key regulators of G protein–coupled receptor 35–arrestin interactions

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

Effects of Electron Radiation on Serotonin Signaling and Reactivity of Rat Gastric Smooth Muscle.

Ionizing radiation in radiotherapy can disrupt cellular functions based on radiation type, energy, and dose. However, investigations on the effects of accelerated electrons, particularly on serotonin mediation, are limited. This study aimed to investigate changes in serotonin signal transduction (targeting 5-HT2A and 5-HT2B receptors) in gastric smooth muscle (SM) samples isolated from rats irradiated with accelerated electrons (linear accelerator Siemens Primus S/N 3561) and their effects on serotonin-induced reactions. The radiation effects were examined in samples prepared five days after the procedure. The contractile activity of smooth muscle samples was measured using an isometric method. The expression of 5-HT2A and 5-HT2B receptors was determined by immunohistochemical assay. Increased contractile reactivity to exogenous serotonin (1.10-8-1.10-4 mol/L) was observed in irradiated samples compared to controls. The expression of 5-HT2A and 5-HT2B receptors was significantly increased in the irradiated tissue. By selecting appropriate time intervals between equimolar (1.10-6 mol/L) sequential serotonin exposures, a process of desensitization associated with agonist-induced internalization was established in control samples, which was absent in irradiated samples. In conclusion, irradiation with accelerated electrons affects the agonist-induced receptor internalization of 5-HT2A and 5-HT2B receptors and increases their expression in rat gastric SM, which alters their contractile reactivity to exogenous serotonin.

A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis

Group I metabotropic glutamate receptors (mGluRs) play important roles in many neuronal processes and are believed to be involved in synaptic plasticity underlying the encoding of experience, including classic paradigms of learning and memory. These receptors have also been implicated in various neurodevelopmental disorders, such as Fragile X syndrome and autism. Internalization and recycling of these receptors in the neuron are important mechanisms to regulate the activity of the receptor and control the precise spatiotemporal localization of these receptors. Applying a "molecular replacement" approach in hippocampal neurons derived from mice, we demonstrate a critical role for protein interacting with C kinase 1 (PICK1) in regulating the agonist-induced internalization of mGluR1. We show that PICK1 specifically regulates the internalization of mGluR1, but it does not play any role in the internalization of the other member of group I mGluR family, mGluR5. Various regions of PICK1 viz., the N-terminal acidic motif, PDZ domain, and BAR domain play important roles in the agonist-mediated internalization of mGluR1. Finally, we demonstrate that PICK1-mediated internalization of mGluR1 is critical for the resensitization of the receptor. Upon knockdown of endogenous PICK1, mGluR1s stayed on the cell membrane as inactive receptors, incapable of triggering the MAP kinase signaling. They also could not induce AMPAR endocytosis, a cellular correlate for mGluR-dependent synaptic plasticity. Thus, this study unravels a novel role for PICK1 in the agonist-mediated internalization of mGluR1 and mGluR1-mediated AMPAR endocytosis that might contribute to the function of mGluR1 in neuropsychiatric disorders.

Biased signaling due to oligomerization of the G protein-coupled platelet-activating factor receptor

G protein-coupled receptors (GPCRs) are important drug targets that mediate various signaling pathways by activating G proteins and engaging β-arrestin proteins. Despite its importance for the development of therapeutics with fewer side effects, the underlying mechanism that controls the balance between these signaling modes of GPCRs remains largely unclear. Here, we show that assembly into dimers and oligomers can largely influence the signaling mode of the platelet-activating factor receptor (PAFR). Single-particle analysis results show that PAFR can form oligomers at low densities through two possible dimer interfaces. Stabilization of PAFR oligomers through cross-linking increases G protein activity, and decreases β-arrestin recruitment and agonist-induced internalization significantly. Reciprocally, β-arrestin prevents PAFR oligomerization. Our results highlight a mechanism involved in the control of receptor signaling, and thereby provide important insights into the relationship between GPCR oligomerization and downstream signaling.

Agonist-Promoted Phosphorylation and Internalization of the Kappa Opioid Receptor in Mouse Brains: Lack of Connection With Conditioned Place Aversion.

Selective kappa opioid receptor (KOR) agonists are promising antipruritic agents and analgesics. However, clinical development of KOR agonists has been limited by side effects, including psychotomimetic effects, dysphoria, and sedation, except for nalfurafine, and recently. CR845 (difelikefalin). Activation of KOR elicits G protein- and β-arrestin-mediated signaling. KOR-induced analgesic and antipruritic effects are mediated by G protein signaling. However, different results have been reported as to whether conditioned place aversion (CPA) induced by KOR agonists is mediated by β-arrestin signaling. In this study, we examined in male mice if there was a connection between agonist-promoted CPA and KOR phosphorylation and internalization, proxies for β-arrestin recruitment in vivo using four KOR agonists. Herein, we demonstrated that at doses producing maximal effective analgesic and antiscratch effects, U50,488H, MOM-SalB, and 42B, but not nalfurafine, promoted KOR phosphorylation at T363 and S369 in mouse brains, as detected by immunoblotting with phospho-KOR-specific antibodies. In addition, at doses producing maximal effective analgesic and antiscratch effects, U50,488H, MOM-SalB, and 42B, but not nalfurafine, caused KOR internalization in the ventral tegmental area of a mutant mouse line expressing a fusion protein of KOR conjugated at the C-terminus with tdTomato (KtdT). We have reported previously that the KOR agonists U50,488H and methoxymethyl salvinorin B (MOM-SalB) cause CPA, whereas nalfurafine and 42B do not, at doses effective for analgesic and antiscratch effects. Taken together, these data reveal a lack of connection between agonist-promoted KOR-mediated CPA with agonist-induced KOR phosphorylation and internalization in male mice.

AKAP12 Modulates β‐arrestin2 Recruitment to the β2‐Adrenergic Receptors

BackgroundG‐Protein Coupled Receptors (GPCRs) are central mediators of signaling cascades in many biological systems. GPCRs internalization rates vary based on the presence/absence of its ligand and the type of the receptor. β2‐Adrenergic Receptor (β2AR) is a prototypical GPCR, reaching its maximum in internalization within ~10 minutes after ligand treatment. One regulator of β2AR internalization is β‐arrestin2. GPCR–β‐arrestin binding is biphasic where the phosphorylated carboxyl terminus of GPCRs docks to the N‐domain of β‐arrestin first, and then the seven transmembrane core of the receptor engages with β‐arrestin. However, it has been discovered that the core interaction is dispensable for receptor endocytosis. AKAP12 is a large scaffolding protein that was reported to be essential in agonist‐induced internalization and resensitization of β2AR. Our central hypothesis is that higher expression levels of AKAP12 may enhance β‐arrestin2 recruitment to the β2AR and subsequently increase β2AR internalization.MethodsHEK293 cells were grouped into three groups: [1] Controls: endogenous AKAP12 expression, [2] Overexpression (OX): transiently overexpressing human AKAP12, and [3] Knockdown (KD): treated with human AKAP12 siRNA. β‐arrestin2 recruitment to the receptor and β2AR internalization was evaluated using the Nanobit luciferase system. For each assay, we tested four β2AR‐β‐arrestin2 or Early Endosome‐β2AR orientations. Orientation pairs with the highest luminescence signals were used for the experiments to obtain maximum sensitivity. At 48 hours post‐transfection, Nanobit assays were performed by recording baseline luminescence for 10 minutes. Next, 10µM epinephrine was added and luminescence measurements were recorded immediately and then once every minute for 1 hour. Results are represented as Luminescence Fold Induction (LFI). Receptor trafficking to early endosomes post epinephrine treatment was further confirmed using the Proximity Ligation Assay (PLA).ResultsHEK cells, OX AKAP12 [9 fold higher] had a significantly lower β‐arrestin2 recruitment to the β2AR (2.26±0.39, LFI) as compared to controls (7.47±1.12, LFI; p=0.0004). AKAP12 KD [AKAP12 ~80% silenced] had a significantly higher β‐arrestin2 recruitment to the β2AR (10.35±0.56, LFI) as compared to both control and OX AKAP12 groups (p=0.035 and p<0.0001), respectively. At 15 minutes post epinephrine addition, β2AR trafficking to early endosomes was significantly lower in the OX AKAP12 group (1.82±0.22, LFI), as compared to the control (2.47±0.09, LFI; p=0.01), while the KD AKAP12 group had a significantly higher internalization (2.98±0.14, LFI), as compared to control and OX AKAP12 groups (p=0.005 and p=0.0003), respectively. Our PLA data further confirmed that the OX AKAP12 group displayed significantly lower β2AR trafficking as defined by its decreased interaction with Early Endosome Antigen 1 (EEA1), as compared to the control (p= 0.0451).ConclusionsContrary to previously thought, higher AKAP12 expression levels may interfere with β2AR trafficking to early endosomes partially through inhibiting β‐arrestin2 recruitment to the β2AR.

Abstract 13074: Impact of Adrenergic Receptor Polymorphisms on Incident Ventricular Fibrillation During Acute Myocardial Ischemia: The MAP-MI Study

Rationale: During acute coronary syndromes (ACS) adrenergic receptor (AR) signaling may have major effects on cardiac arrhythmias including VF. AR gene products are highly polymorphic, and minor variants exhibit potentially important pharmacologic differences compared to receptors encoded by major allele homozygotes ( mah ). Objective: We tested the general hypothesis that polymorphic variants in the ADRB1 , ADRB2 or ADRA2C genes affect incident VF during ACS. Methods and Results: We recruited 953 subjects with a proven MI and no previous history of AC. In a case-control design 477 VF patients were compared to 476 controls without VF who did not differ in baseline characteristics. DNA collection and subsequent genotyping for ADRB1 389/49, ADRB2 27/16 and ADRA2C 322-325 Ins/Del were available in 718-779 patients. Results were expressed as Odds Ratios (OR) for VF vs. No-VF subjects by comparing each mah to minor allele monotype genotypes, genotype combinations or haplotypes. The entire cohort OR (95% CI) for patients receiving (41%) vs. not receiving beta-blockade (BB) was (0.70 (0.53, 0.93), P=0.014), dictating a BB adjustment in the genotype-VF analyses. No minor allele genotype was associated with increased incident VF. Monotypes with statistically significant ORs and their mah comparators were: ADRB2 monotypes and ADRB1 or ADRB2 haplotypes were not associated with VF reduction. Two of 3 ADRB1 and 3 of 4 ADRB2 monotypes or genotype combinations associated with VF reduction encoded ARs that exhibit increased agonist-induced internalization. Conclusions: In acute MI AR variants associated with reduced incident VF are those that mediate increased adrenergic drive ( ADRA2 C Del) and beta-AR internalization. This combination would be predicted to redirect beta-AR signaling from canonical pathways to "biased" EGFR and ERK1/2 mediated cardioprotection.

Functional independence of endogenous μ- and δ-opioid receptors co-expressed in cholinergic interneurons.

Class A G-protein-coupled receptors (GPCRs) normally function as monomers, although evidence from heterologous expression systems suggests that they may sometimes form homodimers and/or heterodimers. This study aims to evaluate possible functional interplay of endogenous µ- and δ-opioid receptors (MORs and DORs) in mouse neurons. Detecting GPCR dimers in native tissues, however, has been challenging. Previously, MORs and DORs co-expressed in transfected cells have been reported to form heterodimers, and their possible co-localization in neurons has been studied in knock-in mice expressing genetically engineered receptors fused to fluorescent proteins. Here, we find that single cholinergic neurons in the mouse striatum endogenously express both MORs and DORs. The receptors on neurons from live brain slices were fluorescently labeled with a ligand-directed labeling reagent, NAI-A594. The selective activation of MORs and DORs, with DAMGO (µ-agonist) and deltorphin (δ-agonist) inhibited spontaneous firing in all cells examined. In the continued presence of agonist, the firing rate returned to baseline as the result of receptor desensitization with the application of deltorphin but was less observed with the application of DAMGO. In addition, agonist-induced internalization of DORs but not MORs was detected. When MORs and DORs were activated simultaneously with [Met5]-enkephalin, desensitization of MORs was facilitated but internalization was not increased. Together, these results indicate that while MORs and DORs are expressed in single striatal cholinergic interneurons, the two receptors function independently.

A Limited or a Transient AT1R Internalization Induced by Two Discriminating Agonists Result in Distinct Downstream AT1R Signaling: ERK1/2 and Akt/PKB

Background Agonist-induced AT1R Internalization, results from sequestration of the agonist-receptor complex into “clathrin vesicles”, depending on the internalization proteins: β-arrestin, clathrin. In this regard, we have shown that a 15,000 kDa polymer of Ang II (Ang II-POL) at maximal concentration, upon a sustained action on the AT1R of rat coronary endothelial luminal membrane (CELM) produced: a sustained inotropic effect, a lack of endothelial AT1R internalization, enhanced membranal recruitment of β-arrestin1/2, without clathrin recruitment a lack of clathrin-vesicles formation. In contrast, the monomer Ang II (1 kDa) inotropic effects shows desensitization, paralleled by endothelial AT1R internalization, and the well-known gradual membranal recruitment of the internalization proteins β-arrestin1/2 and clathrin. These results clearly show that Ang II and Ang II-POL distinctly activate AT1R as reflected by their different recruitment ways of the internalization proteins, which likely result in distinct downstream AT1R signaling: ERK1/2 and Akt/PKB phosphorylation. We studied this canonical signaling pathways triggered by AT1R activation, with both agonists. Hypothesis The distinct rates of membrane-recruitment of the internalization proteins; β-arrestin and clathrin by Ang II and Ang II-POL likely results also in distinct downstream AT1R signaling: ERK1/2, Akt/PKB phosphorylation. Hypothesis test C9 cells were used as a model of cytoplasmic events. Cells were stimulated either with maximal concentrations of Ang II or Ang II-POL and under various conditions were determined the changes in ERK1/2 and Akt/PKB phosphorylation: 1) at various times, 2) in the presence of effect of AT1R and AT2R antagonist and 3) in the presence of various PI3K and MEK1 inhibitors. Results 1) Ang II as well as Ang II-POL caused, via an AT1R activation, a time dependent phosphorylation of ERK1/2 and Akt/PKB. These time-dependent responses, 2) in the case of Akt the response by Ang II was transient while for Ang II-POL rose to a plateau and sustained, but, 3) in the case of ERK1/2’s for both agonists rose followed by a gradual drop and were equally transient. In addition, 4) PI3K inhibitors blocked the Akt responses induced Ang II and Ang II-POL, however, in the case of ERK1/2, the inhibitors only blocked the effects of Ang II-POL but not Ang II's. Conclusion The distinct rates of membrane-recruitment of the internalization proteins; β-arrestin and clathrin by Ang II and Ang II-POL are associated with distinct downstream AT1R signaling: ERK1/2, Akt/PKB. For example, a sustained AT1R activation or recruitments, enhanced for β-arrestin1/2 and inhibited for clathrin prevent desensitization of Akt/PKB; in addition, PI3K dependent phosphorylation of ERK. Clearly, AngII-POL constitutes a powerful tool for understanding the internalization and intracellular signaling mechanisms, induced by sustained AT1R activation.

CRISPR/Cas9‐mediated knockout of G proteins and β‐arrestins defines their specific but interdependent roles in dopamine D1 receptor signaling

The dopamine D1 receptor (D1R) is a G protein-coupled receptor which regulates key brain functions including voluntary movement, memory, attention, and reward. Selective agonist activation of the D1R may also provide novel neurotherapeutics for neurodegenerative and neuropsychiatric disorders. The D1R canonically activates the heterotrimeric G proteins Gαs, and possibly Gαolf, which activate adenylyl cyclase (AC) to increase cAMP/PKA signaling. The D1R also engages β-arrestin proteins which may transduce β-arrestin dependent signaling by scaffolding and activating kinases. However, the distinct roles of Gαs, Gαolf versus β-arrestins 1 and 2 for D1R signaling remains largely undefined. Here we utilize CRISPR/Cas9 engineered HEK293 cells in which Gαs, Gαolf, β-arrestin1 and β-arrestin2 are stably knocked out (KO) to delineate D1R pharmacology and signaling mechanisms. Western blots confirmed a complete loss of Gαs, Gαolf, β-arrestin1 or β-arrestin2 proteins in KO cells versus parental wildtype cells. Dose responses with the D1R agonist SKF-81297 or β2AR agonist isoproterenol induced robust cAMP production in wildtype cells that was eliminated in Gαs/olf KO cells. Re-expression of Gαs or Gαolf in KO cells rescued D1R-mediated cAMP signaling similar to wildtype cells. Interestingly, the D1R had significantly higher cAMP signaling when coupling via Gαs as compared to Gαolf, while the β2AR only coupled with Gαs to stimulate cAMP signaling. Western blotting indicated Gαs/olf KO completely blocked agonist induced CREB phosphorylation by both D1R and β2AR agonists, indicating a strict dependence on Gαs/olf and cAMP/PKA signaling to activate CREB. In major contrast, β-arrestin1/2 KO significantly increased the intensity and duration of cAMP signaling and CREB phosphorylation by the β2AR. β-arrestin1/2 KO also entirely blocked agonist-induced D1R internalization and impaired D1R desensitization while Gαs/olf KO had no effect on D1R internalization. Key controls of re-expressing Gαs, Gαolf, β-arrestin1 or β-arrestin2 in KO cells completely restored signaling or internalization similar to wildtype cells, indicating CRISPR/Cas9 targeting did not result in non-specific or compensatory effects in the pathways under study. Notably, β-arrestin1/2 KO also potentiated and prolonged D1R or β2AR-induced ERK1/2 phosphorylation. D1R-induced ERK1/2 phosphorylation in β-arrestin1/2 KO cells was eliminated by the SRC inhibitor SU6656 or the MEK inhibitor U0126, suggesting that D1R signaling via a Gαs/SRC/MEK pathway is essential for ERK1/2 activation. Taken together, our studies indicate that Gαs is the dominant transducer for D1R cAMP, CREB and ERK1/2 signaling and that β-arrestins provide distinctly interdependent roles with G proteins in modulating the duration and intensity of D1R and β2AR signaling cascades. Using CRISPR/Cas9 KO model cells, future studies will examine the distinct contributions of β-arrestins 1 and 2 and Gαs/olf for D1R pharmacology, which will be useful in the development of novel D1R biased agonists.

The use of fluorescence correlation spectroscopy to monitor cell surface β2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying β2 -adrenoceptor (β2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged β2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged β2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface β2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface β2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting β2AR agonists.

Human MC4R variants affect endocytosis, trafficking and dimerization revealing multiple cellular mechanisms involved in weight regulation

SummaryThe Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, β-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy.

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase.

G protein-biased μ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased μ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced μ receptor desensitisation was probed using kinase inhibitors. Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased μ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular μ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid μ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. Compound 1 is a novel G protein-biased μ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

A simple novel approach for detecting blood-brain barrier permeability using GPCR internalization.

Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2). The clinically approved SST2 agonist octreotide (1kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor. We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB openinginduced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development. This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions.