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G‐protein‐coupled receptors (GPCRs) have been shown to form dimers, but the relevance of this phenomenon in G‐protein activation is not known. Among the large GPCR family, metabotropic glutamate (mGlu) receptors are constitutive dimers. Here we examined whether both heptahelical domains (HDs) are turned on upon full receptor activation. To that aim, we measured G‐protein coupling efficacy of dimeric mGlu receptors in which one subunit bears specific mutations. We show that a mutation in the third intracellular loop (i3 loop) known to prevent G‐protein activation in a single subunit decreases coupling efficacy. However, when a single HD is blocked in its inactive state using an inverse agonist, 2‐methyl‐6‐(phenylethynyl)pyridine (MPEP), no decrease in receptor activity is observed. Interestingly, in a receptor dimer in which the subunit that binds MPEP is mutated in its i3 loop, MPEP enhances agonist‐induced activity, reflecting a ‘better’ activation of the adjacent HD. These data are consistent with a model in which a single HD is turned on upon activation of such homodimeric receptors and raise important issues in deciphering the functional role of GPCR dimer formation for G‐protein activation.

The β₂-adrenergic receptor (β₂AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the β₂AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the β₂AR-Gs complex revealed the interaction interface of β₂AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549–555 (2011)], yet, the dynamic process of the β₂AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the β₂AR in response to the full agonist BI-167107 and Gs and Gi1. These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the β₂AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the β₂AR-Gs and the β₂AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs. These differences between the β₂AR-Gs and β₂AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.

Background and purpose: Most of the pharmaceuticals target G‐protein‐coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin‐releasing factor receptor type 1 (CRF1) ligands. Experimental approach: We systematically substituted urocortin, a natural peptide agonist of CRF1, with bulky amino acids (benzoyl‐phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF1 to Gs‐ and Gi‐protein in human embryonic kidney cells, using receptor binding, [35S]‐GTPγS binding stimulation, and cAMP accumulation assays. Key results: Native ligands stimulated Gs and Gi activation through CRF1, resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6–15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non‐selectively activated Gs and Gi, as urocortin did. Conclusions and implications: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G‐proteins by CRF1. This finding implies significant differences between active conformations of the receptor when coupled to different G‐proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling‐selective drug candidates. British Journal of Pharmacology (2007) 151, 851–859; doi:10.1038/sj.bjp.0707293

Profiles of G protein activation have been assessed using a [35S]‐GTPγS binding/immunoprecipitation strategy in Chinese hamster ovary cells expressing either M1, M2, M3 or M4 muscarinic acetylcholine (mACh) receptor subtypes, where expression levels of M1 and M3, or M2 and M4 receptors were approximately equal. Maximal [35S]‐GTPγS binding to Gq/11α stimulated by M1/M3 receptors, or Gi1 – 3α stimulated by M2/M4 receptors occurred within approximately 2 min of agonist addition. The increases in Gq/11α‐[35S]‐GTPγS binding after M1 and M3 receptor stimulation differed substantially, with M1 receptors causing a 2 – 3 fold greater increase in [35S]‐GTPγS binding and requiring 5 fold lower concentrations of methacholine to stimulate a half‐maximal response. Comparison of M2 and M4 receptor‐mediated Gi1 – 3α‐[35S]‐GTPγS binding also revealed differences, with M2 receptors causing a greater increase in Gi1 – 3α activation and requiring 10 fold lower concentrations of methacholine to stimulate a half‐maximal response. Comparison of methacholine‐ and pilocarpine‐mediated effects revealed that the latter partial agonist is more effective in activating Gi3α compared to Gi1/2α for both M2 and M4 receptors. More marked agonist/partial agonist differences were observed with respect to M1/M3‐mediated stimulations of Gq/11α‐ and Gi1 – 3α‐[35S]‐GTPγS binding. Whereas coupling to these Gα subclasses decreased proportionately for M1 receptor stimulation by these agonists, pilocarpine possesses a greater intrinsic activity at M3 receptors for Giα versus Gq/11α activation. These data demonstrate that mACh receptor subtype and the nature of the agonist used govern the repertoire of G proteins activated. They also provide insights into how the diversity of coupling can be pharmacologically exploited, and provide a basis for a better understanding of how multiple receptor subtypes can be differentially regulated. British Journal of Pharmacology (2001) 132, 950–958; doi:10.1038/sj.bjp.0703892

Biased signaling or functional selectivity occurs when a 7TM-receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor), different receptors (with the same ligand), or different tissues or cells (for the same ligand-receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may not be absolute, i.e., full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro-switches based on recently published 7TM crystals and molecular dynamics studies. All three forms of biased signaling are abundant in the chemokine system. This challenges our understanding of \"classic\" redundancy inevitably ascribed to this system, where multiple chemokines bind to the same receptor and where a single chemokine may bind to several receptors - in both cases with the same functional outcome. The ubiquitous biased signaling confers a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles and different functional outcomes in a ligand-, receptor-, or cell/tissue-defined manner. As the low number of successful drug development plans implies, there are great difficulties in targeting chemokine receptors; in particular with regard to receptor antagonists as anti-inflammatory drugs. Un-defined and putative non-selective targeting of the complete cellular signaling system could be the underlying cause of lack of success. Therefore, biased ligands could be the solution.

Background and purpose: According to the two‐domain model for the corticotropin‐releasing factor receptor type 1 (CRF1), peptide antagonists bind to the N‐terminal domain (N‐domain), non‐peptide antagonists to the transmembrane region (J‐domain), whereas peptide agonists attach to both the N‐ and J‐domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs‐ and Gi‐protein coupling of CRF1 by a peptide (α‐helical CRF(9–41)) and non‐peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF1 states for Gs and Gi coupling are similar or different. Experimental approach: We studied the inhibitory effect of α‐helical CRF(9–41) and antalarmin on the coupling of CRF1 to Gs‐ and Gi‐protein in human embryonic kidney cells, using the [35S]‐GTPγS binding stimulation assay. Key results: The non‐peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin‐ and sauvagine‐evoked G‐protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism. Conclusions and Implications: The results allow us to extend the two‐domain model of CRF1 activation by assuming that CRF1 agonists activate the receptor by binding to at least two ensembles of J‐domain configurations which couple to Gs or Gi, that are in turn antagonized by a non‐peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non‐peptide antagonism is not valid for the Gs‐mediated physiological activities of CRF1. British Journal of Pharmacology (2006) 149, 942–947. doi:10.1038/sj.bjp.0706926

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R–mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R–mGsi and –mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.

G protein-coupled receptor kinases (GRKs) constitute seven subtypes of serine/threonine protein kinases that specifically recognize and phosphorylate agonist-activated G protein-coupled receptors (GPCRs), thereby terminating the GPCRs-mediated signal transduction pathway. Recent research shows that GRKs also interact with non-GPCRs and participate in signal transduction in non-phosphorylated manner. Besides, GRKs activity can be regulated by multiple factors. Changes in GRKs expression have featured prominently in various tumor pathologies, and they are associated with angiogenesis, proliferation, migration, and invasion of malignant tumors. As a result, GRKs have been intensively studied as potential therapeutic targets. Herein, we review evolving understanding of the function of GRKs, the regulation of GRKs activity and the role of GRKs in human malignant tumor pathophysiology.

Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D3 receptors (D3Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D3R and either MT1 (MT1R) or MT2 (MT2R) melatonin receptors. Heteromerization led to the blockade of D3R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D3R–MT1R and D3R–MT1R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.

Our data indicate the significant intrinsic efficacy of GABAB-receptors in rat brain cortex already at birth (PD1, PD2). Subsequently, baclofen- and SKF97541-stimulated G-protein activity, measured by agonist-stimulated, high-affinity [35S]GTPγS binding assay, was increased; the highest level of both baclofen and SKF97541-stimulated [35S]GTPγS binding was detected between PD10 and PD15. In older rats, baclofen- and SKF97541-stimulated [35S]GTPγS binding was continuously decreased so, that the level in adult, 90-days old animals, was not different from that in newborn animals. The potency of G-protein response to baclofen (characterized by EC50 values) was also high at birth but unchanged by further postnatal development. An individual variance among different agonists was observed in this respect as the potency of SKF97541 response was decreased between the birth and adulthood. Accordingly, the highest plasma membrane density of GABAB-R, determined by saturation binding assay with antagonist [3H]CGP54626, was measured in 1-day old animals (2.27±0.08 pmol · mg-1). The further development was reflected in a decrease of [3H]CGP54626 binding as the Bmax values of 1.38±0.05 and 0.93±0.04 pmol · mg-1 were determined in PM isolated from 13- and 90-days old rats, respectively.