Orphan GPCR GPRC5C Facilitates Angiotensin II-Induced Smooth Muscle Contraction.

BACKGROUND: GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. METHODS: We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. RESULTS: Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. CONCLUSIONS: Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.

Correction: Al-Zamel, N., et al. A Dual GLP-1/GIP Receptor Agonist Does Not Antagonize Glucagon at Its Receptor but May Act as a Biased Agonist at the GLP-1 Receptor. Int. J. Mol. Sci. 2019, 20, 3532.

The author wishes to make the following correction to this paper [...].

The N-termini of GRK2 and GRK3 simulate the stimulating effects of RKIP on ß-adrenoceptors.

The Raf kinase inhibitor protein (RKIP) activates ß-adrenoceptors (ß-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated ß-AR activation, chronic activation of ß-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated ß-AR phosphorylation and desensitization. Here, we evaluate RKIP-mediated effects on ß-AR to explore new strategies for ß-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 - not GRK5 - as well as several RKIP binding sites within their N-termini (GRK21-185 and GRK31-185). Overexpression of these N-termini prevented ß2-AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of ß2-AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction. Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on ß-AR signaling in HEK293 cells and in cardiomyocytes by their binding to ß2-AR and, thus, provide important insights for the development of new strategies to modulate ß2-AR signaling.

A Dual GLP-1/GIP Receptor Agonist Does Not Antagonize Glucagon at Its Receptor but May Act as a Biased Agonist at the GLP-1 Receptor.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of metabolism, making their receptors (GLP-1R and GIPR) attractive targets in the treatment of type 2 diabetes mellitus (T2DM). GLP-1R agonists are used clinically to treat T2DM but the use of GIPR agonists remains controversial. Recent studies suggest that simultaneous activation of GLP-1R and GIPR with a single peptide provides superior glycemic control with fewer adverse effects than activation of GLP-1R alone. We investigated the signaling properties of a recently reported dual-incretin receptor agonist (P18). GLP-1R, GIPR, and the closely related glucagon receptor (GCGR) were expressed in HEK-293 cells. Activation of adenylate cyclase via Gαs was monitored using a luciferase-linked reporter gene (CRE-Luc) assay. Arrestin recruitment was monitored using a bioluminescence resonance energy transfer (BRET) assay. GLP-1, GIP, and glucagon displayed exquisite selectivity for their receptors in the CRE-Luc assay. P18 activated GLP-1R with similar potency to GLP-1 and GIPR with higher potency than GIP. Interestingly, P18 was less effective than GLP-1 at recruiting arrestin to GLP-1R and was inactive at GCGR. These data suggest that P18 can act as both a dual-incretin receptor agonist, and as a G protein-biased agonist at GLP-1R.

Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling.

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489-Ser495 and Ser501-Thr506) specifically responsible for the majority of PTH(1-34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1-34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489-Ser495 and the second cluster Ser501-Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.

Influence of gαq on the dynamics of m3-acetylcholine receptor-g-protein-coupled receptor kinase 2 interaction.

G-protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase with an important function in the desensitization of G-protein-coupled receptors. Based on its ability to bind G-protein ßγ subunits as well as activated Gαq subunits, it can be considered as an effector for G-proteins. The recruitment of GRK2 to activated receptors is well known to be mediated by Gßγ together with negatively charged membrane phospholipids. In the current study, we address the role of Gαq on the interaction of GRK2 with activated Gq-protein-coupled receptors. Therefore, we established new Förster resonance energy transfer (FRET)-based assays to study the interaction of GRK2 with the M3-acetylcholine (M3-ACh) receptor as well as Gq-protein subunits with high spatiotemporal resolution in single living human embryonic kidney 293T cells. M3-ACh receptor stimulation with 10 µM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding affinity toward Gαq [GRK2(D110A)] and Gßγ [GRK2(R587Q)] were used to determine the specific role of Gq-protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Gαq enhances the extent and stability of the GRK2-M3-ACh receptor interaction, and that not only Gßγ but also Gαq can target GRK2 to the membrane. This reveals an important role of Gαq in efficient recruitment of GRK2 to M3-ACh receptors. Furthermore, interactions between Gαq and GRK2 were associated with a prolongation of the interaction between GRK2 and the M3-ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Gαq influences signaling and desensitization.

Engineered hyperphosphorylation of the ß2-adrenoceptor prolongs arrestin-3 binding and induces arrestin internalization.

G protein-coupled receptor phosphorylation plays a major role in receptor desensitization and arrestin binding. It is, however, unclear how distinct receptor phosphorylation patterns may influence arrestin binding and subsequent trafficking. Here we engineer phosphorylation sites into the C-terminal tail of the ß2-adrenoceptor (ß2AR) and demonstrate that this mutant, termed ß2AR(SSS), showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. By measuring arrestin-3 recruitment and the stability of arrestin-3 receptor complexes in real time using fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that arrestin-3 dissociated quickly and almost completely from the ß2AR, whereas the interaction with ß2AR(SSS) was 2- to 4-fold prolonged. In contrast, arrestin-3 interaction with a ß2-adrenoceptor fused to the carboxyl-terminal tail of the vasopressin type 2 receptor was nearly irreversible. Further analysis of arrestin-3 localization revealed that by engineering phosphorylation sites into the ß2-adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes after internalization. This is in contrast to the wild-type receptor that interacts transiently with arrestin-3 at the plasma membrane. Furthermore, ß2AR(SSS) internalized more efficiently than the wild-type receptor, whereas recycling was very similar for both receptors. Thus, we show how the interaction between arrestins and receptors can be increased with minimal receptor modification and that relatively modest increases in receptor-arrestin affinity are sufficient to alter arrestin trafficking.

Potassium channel TASK-5 forms functional heterodimers with TASK-1 and TASK-3 to break its silence.

TASK-5 (KCNK15) belongs to the acid-sensitive subfamily of two-pore domain potassium (K2P) channels, which includes TASK-1 and TASK-3. TASK-5 stands out as K2P channel for which there is no functional data available, since it was reported in 2001 as non-functional and thus "silent". Here we show that TASK-5 channels are indeed non-functional as homodimers, but are involved in the formation of functional channel complexes with TASK-1 and TASK-3. TASK-5 negatively modulates the surface expression of TASK channels, while the heteromeric TASK-5-containing channel complexes located at the plasma membrane are characterized by changes in single-channel conductance, Gq-coupled receptor-mediated channel inhibition, and sensitivity to TASK modulators. The unique pharmacology of TASK-1/TASK-5 heterodimers, affected by a common polymorphism in KCNK15, needs to be carefully considered in the future development of drugs targeting TASK channels. Our observations provide an access to study TASK-5 at the functional level, particularly in malignant cancers associated with KCNK15.

Endomorphin-2: a biased agonist at the µ-opioid receptor.

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the µ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

Residency time of agonists does not affect the stability of GPCR-arrestin complexes.

BACKGROUND AND PURPOSE: The interaction of arrestins with G-protein coupled receptors (GPCRs) desensitizes agonist-dependent receptor responses and often leads to receptor internalization. GPCRs that internalize without arrestin have been classified as "class A" GPCRs whereas "class B" GPCRs co-internalize with arrestin into endosomes. The interaction of arrestins with GPCRs requires both agonist activation and receptor phosphorylation. Here, we ask the question whether agonists with very slow off-rates can cause the formation of particularly stable receptor-arrestin complexes. EXPERIMENTAL APPROACH: The stability of GPCR-arrestin-3 complexes at two class A GPCRs, the ß2 -adrenoceptor and the µ opioid receptor, was assessed using two different techniques, fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) employing several ligands with very different off-rates. Arrestin trafficking was determined by confocal microscopy. KEY RESULTS: Upon agonist washout, GPCR-arrestin-3 complexes showed markedly different dissociation rates in single-cell FRET experiments. In FRAP experiments, however, all full agonists led to the formation of receptor-arrestin complexes of identical stability whereas the complex between the µ receptor and arrestin-3 induced by the partial agonist morphine was less stable. Agonists with very slow off-rates could not mediate the co-internalization of arrestin-3 with class A GPCRs into endosomes. CONCLUSIONS AND IMPLICATIONS: Agonist off-rates do not affect the stability of GPCR-arrestin complexes but phosphorylation patterns do. Our results imply that orthosteric agonists are not able to pharmacologically convert class A into class B GPCRs.

Differential effects of glucose-dependent insulinotropic polypeptide receptor/glucagon-like peptide-1 receptor heteromerization on cell signaling when expressed in HEK-293 cells.

The incretin hormones: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important regulators of many aspects of metabolism including insulin secretion. Their receptors (GIPR and GLP-1R) are closely related members of the secretin class of G-protein-coupled receptors. As both receptors are expressed on pancreatic ß-cells there is at least the hypothetical possibility that they may form heteromers. In the present study, we investigated GIPR/GLP-1R heteromerization and the impact of GIPR on GLP-1R-mediated signaling and vice versa in HEK-293 cells. Real-time fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) saturation experiments confirm that GLP-1R and GIPR form heteromers. Stimulation with 1 µM GLP-1 caused an increase in both FRET and BRET ratio, whereas stimulation with 1 µM GIP caused a decrease. The only other ligand tested to cause a significant change in BRET signal was the GLP-1 metabolite, GLP-1 (9-36). GIPR expression had no significant effect on mini-Gs recruitment to GLP-1R but significantly inhibited GLP-1 stimulated mini-Gq and arrestin recruitment. In contrast, the presence of GLP-1R improved GIP stimulated mini-Gs and mini-Gq recruitment to GIPR. These data support the hypothesis that GIPR and GLP-1R form heteromers with differential consequences on cell signaling.

ß-arrestin1 and 2 exhibit distinct phosphorylation-dependent conformations when coupling to the same GPCR in living cells.

ß-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR-ß-arrestin complex. However, whether ß-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and ß-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations ("hanging" and "core"). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of ß-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess ß-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function.

Disentangling bias between Gq, GRK2, and arrestin3 recruitment to the M3 muscarinic acetylcholine receptor.

G protein-coupled receptors (GPCRs) transmit extracellular signals to the inside by activation of intracellular effector proteins. Different agonists can promote differential receptor-induced signaling responses - termed bias - potentially by eliciting different levels of recruitment of effector proteins. As activation and recruitment of effector proteins might influence each other, thorough analysis of bias is difficult. Here, we compared the efficacy of seven agonists to induce G protein, G protein-coupled receptor kinase 2 (GRK2), as well as arrestin3 binding to the muscarinic acetylcholine receptor M3 by utilizing FRET-based assays. In order to avoid interference between these interactions, we studied GRK2 binding in the presence of inhibitors of Gi and Gq proteins and analyzed arrestin3 binding to prestimulated M3 receptors to avoid differences in receptor phosphorylation influencing arrestin recruitment. We measured substantial differences in the agonist efficacies to induce M3R-arrestin3 versus M3R-GRK2 interaction. However, the rank order of the agonists for G protein- and GRK2-M3R interaction was the same, suggesting that G protein and GRK2 binding to M3R requires similar receptor conformations, whereas requirements for arrestin3 binding to M3R are distinct.

µ-opioid receptors: correlation of agonist efficacy for signalling with ability to activate internalization.

We have compared the ability of a number of µ-opioid receptor (MOPr) ligands to activate G proteins with their abilities to induce MOPr phosphorylation, to promote association of arrestin-3 and to cause MOPr internalization. For a model of G protein-coupled receptor (GPCR) activation where all agonists stabilize a single active conformation of the receptor, a close correlation between signaling outputs might be expected. Our results show that overall there is a very good correlation between efficacy for G protein activation and arrestin-3 recruitment, whereas a few agonists, in particular endomorphins 1 and 2, display apparent bias toward arrestin recruitment. The agonist-induced phosphorylation of MOPr at Ser(375), considered a key step in MOPr regulation, and agonist-induced internalization of MOPr were each found to correlate well with arrestin-3 recruitment. These data indicate that for the majority of MOPr agonists the ability to induce receptor phosphorylation, arrestin-3 recruitment, and internalization can be predicted from their ability as agonists to activate G proteins. For the prototypic MOPr agonist morphine, its relatively weak ability to induce MOPr internalization can be explained by its low agonist efficacy.

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro.

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

The Effect of Cell Surface Expression and Linker Sequence on the Recruitment of Arrestin to the GIP Receptor.

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptor are important targets in the treatment of both type 2 diabetes mellitus (T2DM) and obesity. Originally identified for their role in desensitization, internalization and recycling of G protein-coupled receptors (GPCRs), arrestins have since been shown to act as scaffolding proteins that allow GPCRs to signal in a G protein-independent manner. While GLP-1R has been reported to interact with arrestins, this aspect of cell signaling remains controversial for GIPR. Using a (FRET)-based assay we have previously shown that yellow fluorescent protein (YFP)-labeled GIPR does not recruit arrestin. This GIPR-YFP construct contained a 10 amino acid linker between the receptor and a XbaI restriction site upstream of the YFP. This linker was not present in the modified GIPR-SYFP2 used in subsequent FRET and bioluminescence resonance energy transfer (BRET) assays. However, its removal results in the introduction of a serine residue adjacent to the end of GIPR's C-terminal tail which could potentially be a phosphorylation site. The resulting receptor was indeed able to recruit arrestin. To find out whether the serine/arginine (SR) coded by the XbaI site was indeed the source of the problem, it was substituted with glycine/glycine (GG) by site-directed mutagenesis. This substitution abolished arrestin recruitment in the BRET assay but only significantly reduced it in the FRET assay. In addition, we show that the presence of a N-terminal FLAG epitope and influenza hemagglutinin signal peptide were also required to detect arrestin recruitment to the GIPR, most likely by increasing receptor cell surface expression. These results demonstrate how arrestin recruitment assay configuration can dramatically alter the result. This becomes relevant when drug discovery programs aim to identify ligands with "biased agonist" properties.

Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.

Biased agonism at G protein-coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus ß-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as ß-arrestin recruitment. At the µ-opioid receptor (MOR), G protein-biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in ß-arrestin-mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and ß-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target.

Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid µ-opioid receptor desensitization.

G protein receptor kinases (GRKs) and ß-arrestins are key regulators of µ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser375 and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and ß-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was ß-arrestin to a DAMGO-activated MOR. ß-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the 370TREHPSTANT379 motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and ß-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.

Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.

Hormones and neurotransmitters transduce signals through G protein-coupled receptors (GPCR). Despite their common signaling pathways, however, the responses they elicit have different temporal patterns. To reveal the molecular basis for these differences we have developed a generally applicable fluorescence-based technique for real-time monitoring of the activation switch of GPCRs in living cells. We used such direct measurements to investigate the activation of the alpha(2A)-adrenergic receptor (alpha(2A)AR; neurotransmitter) and the parathyroid hormone receptor (PTHR; hormone) and observed much faster kinetics than expected: approximately 40 ms for the alpha(2A)AR and approximately 1 s for the PTHR. The different switch times are in agreement with the different receptors' biological functions. Agonists and antagonists could rapidly switch the receptors on or off, whereas a partial agonist caused only a partial signal. This approach allows the comparison of agonist and partial agonist intrinsic activities at the receptor level and provides evidence for millisecond activation times of GPCRs.