Claudio Miguel Costa-Neto

G protein-coupled receptors (GPCRs) are central to pathophysiological processes and remain prominent targets in drug discovery. Recent advances in understanding GPCR signaling and modulation, such as biased agonism, dual agonism, and non-canonical G protein signaling, have expanded the therapeutic landscape of these receptors. These understandings have led (and are leading further) to innovative approaches that broaden GPCRs as therapeutic targets, going after better efficacy and minimizing adverse effects. However, tachyphylaxis, a rapid decrease in receptor responsiveness after repeated stimulation, presents a significant challenge in a chronic treatment context. Recent findings from our group revealed that tachyphylaxis in the angiotensin type 1 (AT1) receptor is primarily governed by the ligand’s dissociation rate (koff), i.e. high residence time, rather than by β-arrestin-mediated desensitization, as could be expected. This suggests that internalized AT1 receptors remain active when bound to ligands with high residence time, favoring sustained signaling from endosomes. Importantly, the concept of high residence time sheds new light on intracellular signaling phenomena and underscores the therapeutic value of modulating intracellular receptor activity, including the development of novel cell-permeant antagonists. This review discusses critical pharmacological parameters for drug discovery focused on agonists, including (i) activation of preferential signaling pathways (biased agonism), (ii) internalization/recycling rates, (iii) tachyphylaxis/desensitization, (iv) allosteric modulators, and (v) intracellular receptor signaling and its blockade, emphasizing the need for strategies that extend beyond conventional GPCRs’ functional assays. Additionally, this review highlights how advancements in high-resolution imaging, bioluminescence resonance energy transfer-based biosensors, and computational modeling are crucial for elucidating complex GPCRs’ behaviors, particularly in understanding mechanisms like tachyphylaxis and its interplay with compartment-specific signaling. These approaches not only pave the way for therapies that strategically leverage or mitigate tachyphylaxis to sustain receptor responsiveness, but could enable the design of drugs targeting intracellular pathways as a strategy to enhance efficacy and minimize adverse effects. These insights underscore the importance of integrating diverse pharmacological strategies to refine GPCR-targeted therapies and address unmet medical needs, particularly in chronic conditions where sustained receptor activity is critical.

The canonical model of G protein–coupled receptor (GPCR) signaling comprises G protein activation at the plasma membrane, followed by receptor phosphorylation and β-arrestin recruitment, which leads to receptor desensitization and endocytosis. However, the activation of some GPCRs results in sustained G protein signaling from intracellular compartments in a manner reportedly dependent on β-arrestin and receptor endocytosis. The vasopressin type 2 receptor (V ₂ R) can be activated by two structurally similar hormones, arginine vasopressin and oxytocin, both of which stimulate the production of the second messenger cyclic adenosine monophosphate (cAMP). In this study, we showed that sustained V ₂ R signaling and endosomal Gα _s (stimulatory G protein alpha subunit) translocation could occur without β-arrestin–mediated receptor endocytosis and was primarily controlled by the residence time of the ligand on the receptor. β-Arrestin had opposing effects on sustained signaling: It facilitated receptor internalization into endosomes, where it activated Gα _s , and promoted cAMP production from this compartment. However, β-arrestin–mediated receptor endocytosis also induced ligand dissociation due to the acidic endosomal environment, thereby limiting the signal. Overall, our data suggest that signals originating at the plasma membrane play a dominant role in sustained V ₂ R signaling stimulated by arginine vasopressin.

Several GPCRs (G-protein–coupled receptors) have been reported to exhibit tachyphylaxis, which is an acute loss of functional receptor response after repeated stimuli with an agonist. GPCRs are important clinical targets for a wide range of disorders. Therefore, elucidation of the ligand features that contribute to receptor tachyphylaxis and signaling events underlying this phenomenon is important for drug discovery and development. In this study, we examined the role of ligand-binding kinetics in the tachyphylaxis of AT ₁ R (angiotensin II type 1 receptor) using bioluminescence resonance energy transfer assays to monitor signaling events under both kinetic and equilibrium conditions. We investigated AT ₁ R signal transduction and translocation promoted by the endogenous tachyphylactic agonist Ang II (angiotensin II) and its analogs, described previously for inducing reduced receptor tachyphylaxis. Estimation of binding kinetic parameters of the ligands revealed that the residence time of Ang II was higher than that of the analogs, resulting in more sustained G _q protein activation and recruitment of β-arrestin than that promoted by the analogs. Furthermore, we observed that Ang II led to more sustained internalization of the receptor, thereby retarding its recycling to the plasma membrane and preventing further receptor responses. These results show that the apparent lack of tachyphylaxis in the studied analogs resulted from their short residence time at the AT ₁ R. In addition, our data highlight the relevance of complete characterization of novel GPCR drug candidates, taking into account their receptor binding kinetics as well.

Significance: G protein–coupled receptors (GPCRs) have been proposed to signal in multiple compartments within the cell, a phenomenon that could have important implications for our understanding of receptor pharmacology and disease treatment. However, directly measuring G protein activity in subcellular compartments remains a challenge, and the mechanism underlying their translocation and activation remains elusive. Using a biosensor that specifically measures the presence of GTP-loaded G _q/11 at endosomes, our findings reveal the mechanism underlying G _q trafficking and signaling in the endosome and provide a powerful tool to directly monitor G protein movement and activation in subcellular compartments.

The AT1 receptor (AT1R) has a major role in the Renin-Angiotensin System, being involved in several physiological events including blood pressure control and electrolyte balance. The AT1R is a member of the G protein coupled receptors (GPCR) family, classically known to couple G_αqand engage β-arrestin recruitment. Both G protein and arrestin signaling pathways are involved in modulation of different downstream kinases. A previous study reported that mutations in the AT1R (A244S and I103T-A244S) were positively correlated with higher risk of atrial fibrillation in men. Based on that report, we aimed to investigate if these mutations, including I103T only, could affect AT1R signal transduction profile, and consequently, implicate in atrial fibrillation outcome. To address that, we engineered an AT1R carrying the above-mentioned mutations, and functionally evaluated different signaling pathways. Phosphokinase profiler array to assess the mutations downstream effects on kinases and kinase substrates phosphorylation levels was used. Our results show that the I103T-A244S mutant receptor presents decreased β-arrestin 2 recruitment, which could lead to a harmful condition of sustained G_αqsignaling. Moreover, the phosphokinase profiler array revealed that the same mutation led to downstream modulation of kinase pathways that are linked to physiological responses such as fibrous tissue formation, apoptosis and cell proliferation.

We have previously reported that angiotensin II receptor type 1 (AT1R) contributes to the hypertrophic effects of thyroid hormones (TH) in cardiac cells. Even though evidence indicates crosstalks between TH and AT1R, the underlying mechanisms are poorly understood. Beta‐arrestin (ARRB) signaling has been described as noncanonical signal transduction pathway that exerts important effects in the cardiovascular system through G‐protein‐coupled receptors, as AT1R. Herein, we investigated the contribution of ARRB signaling in TH‐induced cardiomyocyte hypertrophy. Primary cardiomyocyte cultures were treated with Triiodothyronine (T3) to induce cell hypertrophy. T3 rapidly activates extracellular signal‐regulated kinase 1/2 (ERK1/2) signaling, which was partially inhibited by AT1R blockade. Also, ERK1/2 inhibition attenuated the hypertrophic effects of T3. ARRB2 was upregulated by T3, and small interfering RNA assays revealed the role of ARRB2—but not ARRB1—on ERK1/2 activation and cardiomyocyte hypertrophy. Corroborating these findings, the ARRB2‐overexpressed cells showed increased expression of hypertrophic markers, which were attenuated by ERK1/2 inhibition. Immunocytochemistry and immunoprecipitation assays revealed the increased expression of nuclear AT1R after T3 stimulation and the increased interaction of AT1R/ARRB2. The inhibition of endocytosis also attenuated the T3 effects on cardiac cells. Our results evidence the contribution of ARRB2 on ERK1/2 activation and cardiomyocyte hypertrophy induced by T3 via AT1R.

Metastatic melanoma is an aggressive type of skin cancer leading half of the patients to death within 8–10 months after diagnosis. Kinins are peptides that interact with B1 and B2 receptors playing diverse biological roles. We investigated whether treatment with B1 receptor agonist, des-Arg⁹-bradykinin (DABK), has effects in lung metastasis establishment after melanoma induction in mice. We found a lower number of metastatic colonies in lungs of DABK-treated mice, reduced expression of vascular cell adhesion molecule 1 (VCAM-1), and increased CD8⁺T-cell recruitment to the metastatic area compared to animals that did not receive treatment. To understand whether the effects of DABK observed were due to the activation of the B1 receptor in the tumor cells or in the host, we treated wild-type (WT) and kinin B1 receptor knockout (B1^−/−) mice with DABK. No significant differences in the number of melanoma colonies established in lungs were seen between WT and B1^−/−mice; however, B1^−/−mice presented higher VCAM-1 expression and lower CD8⁺T-cell infiltration. In conclusion, we believe that activation of kinin B1 receptor by its agonist in the host stimulates the immune response more efficiently, promoting CD8⁺T-cell recruitment to the metastatic lungs and interfering in VCAM-1 expression. Moreover, treatment with DABK reduced establishment of metastatic colonies by mainly acting on tumor cells; hence, this study brings insights to explore novel approaches to treat metastatic melanoma targeting the B1 receptor.

Platyrrhini (New World monkeys, NWm) are a group of primates characterized by behavioral and reproductive traits that are otherwise uncommon among primates, including social monogamy, direct paternal care, and twin births. As a consequence, the study of Platyrrhine primates is an invaluable tool for the discovery of the genetic repertoire underlying these taxon‐specific traits. Recently, high conservation of vasopressin (AVP) sequence, in contrast with high variability of oxytocin (OXT), has been described in NWm. AVP and OXT functions are possible due to interaction with their receptors: AVPR1a, AVPR1b, AVPR2, and OXTR; and the variability in this system is associated with the traits mentioned above. Understanding the variability in the receptors is thus fundamental to understand the function and evolution of the system as a whole. Here we describe the variability of AVPR1b coding region in 20 NWm species, which is well‐known to influence behavioral traits such as aggression, anxiety, and stress control in placental mammals. Our results indicate that 4% of AVPR1b sites may be under positive selection and a significant number of sites under relaxed selective constraint. Considering the known role of AVPR1b, we suggest that some of the changes described here for the Platyrrhini may be a part of the genetic repertoire connected with the complex network of neuroendocrine mechanisms of AVP–OXT system in the modulation of the HPA axis. Thus, these changes may have promoted the emergence of social behaviors such as direct paternal care in socially monogamous species that are also characterized by small body size and twin births.

LASSBio-579, an N-phenylpiperazine antipsychotic lead compound, has been previously reported as a D₂ receptor (D₂R) ligand with antipsychotic-like activities in rodent models of schizophrenia. In order to better understand the molecular mechanism of action of LASSBio-579 and of its main metabolite, LQFM 037, we decided to address the hypothesis of functional selectivity at the D₂R. HEK-293T cells transiently coexpressing the human long isoform of D₂ receptor (D₂LR) and bioluminescence resonance energy transfer (BRET)-based biosensors were used. The antagonist activity was evaluated using different concentrations of the compounds in the presence of a submaximal concentration of dopamine (DA), after 5 and 20 min. For both signaling pathways, haloperidol, clozapine, and our compounds act as DA antagonists in a concentration-dependent manner, with haloperidol being by far the most potent, consistent with its nanomolar D₂R affinity measured in binding assays. In our experimental conditions, only haloperidol presented a robust functional selectivity, being four- to fivefold more efficient for inhibiting translocation of β-arrestin-2 (β-arr2) than for antagonizing Gi activation. Present data are the first report on the effects of LASSBio-579 and LQFM 037 on the β-arr2 signaling pathway and further illustrate that the functional activity could vary depending on the assay conditions and approaches used.