Atypical nuclear localization of VIP receptors in glioma cell lines and patients

An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.     

Vasoactive intestinal polypeptide type-1 receptor regulation. Desensitization, phosphorylation, and sequestration

The vasoactive intestinal polypeptide type-1 (VPAC(1)) receptor is a class II G protein-coupled receptor, distinct from the adrenergic receptor superfamily. The mechanisms involved in the regulation of the VPAC(1) receptor are largely unknown. We examined agonist-dependent VPAC(1) receptor signaling, phosphorylation, desensitization, and sequestration in human embryonic kidney 293 cells. Agonist stimulation of cells overexpressing this receptor led to a dose-dependent increase in cAMP that peaked within 5-10 min and was completely desensitized after 20 min. Cells cotransfected with the VPAC(1) receptor (VPAC(1)R) and G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6 exhibited enhanced desensitization that was not evident with GRK 4. Immunoprecipitation of the epitope-tagged VPAC(1) receptor revealed dose-dependent phosphorylation that was increased with cotransfection of any GRK. Agonist-stimulated internalization of the VPAC(1)R peaked in 10 min, and neither overexpressed beta-arrestin nor its dominant-negative mutant altered internalization. However, a dynamin-dominant negative mutant did inhibit VPAC(1) receptor internalization. Interestingly, VPAC(1)R specificity in desensitization was not evident by study of the overexpressed receptor; however, we determined that human embryonic kidney 293 cells express an endogenous VPAC(1)R that did demonstrate dose-dependent GRK specificity. Therefore, VPAC(1) receptor regulation involves agonist-stimulated, GRK-mediated phosphorylation, beta-arrestin translocation, and dynamin-dependent receptor internalization. Moreover, study of endogenously expressed receptors may provide information not evident in overexpressed systems.     

A Fluorescent Reporter Assay for the Detection of Ligands Acting Through GI Protein-Coupled Receptors

Abstract

Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for G, protein-coupled receptors. Two G_i-GPCRs, μ-opioid receptor (μ-OPR) and 5-hydroxytryptamine receptor la (5HTlaR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric G_q/i5 protein (which re-directs a negative G_i-type signal to a positive Gq-type response), (2) a given G_i-GPCR, and (3) a β-lactamase (βla) reporter gene responsive to G_i-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.     

Design and biological assessment of membrane-tethering neuroprotective peptides derived from the pituitary adenylate cyclase-activating polypeptide type 1 receptor

BackgroundThe pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1), a class B G protein-coupled receptor (GPCR), has emerged as a promising target for treating neurodegenerative conditions. Unfortunately, despite years of research, no PAC1-specific agonist has been discovered, as activity on two other GPCRs, VPAC1 and VPAC2, is retained with current analogs. Cell signaling is related to structural modifications in the intracellular loops (ICLs) of GPCRs. Thus, we hypothesized that peptides derived from the ICLs (called pepducins) of PAC1 might initiate, as allosteric ligands, signaling cascades after recognition of the parent receptor and modulation of its conformational landscape.MethodsThree pepducins were synthesized and evaluated for their ability to 1) promote cell survival; 2) stimulate various signaling pathways associated with PAC1 activation; 3) modulate selectively PAC1, VPAC1 or VPAC2 activation; and 4) sustain mobility and prevent death of dopaminergic neurons in a zebrafish model of neurodegeneration.ResultsAssays demonstrated that these molecules promote SH-SY5Y cell survival, a human neuroblastoma cell line expressing PAC1, and activate signaling via Gα_s and Gα_q, with distinct potencies and efficacies. Also, PAC1-Pep1 and PAC1-Pep2 activated selectively PAC1-mediated Gα_s stimulation. Finally, experiments, using a zebrafish neurodegeneration model, showed a neuroprotective action with all three pepducins and in particular, revealed the ability of PAC1-Pep1 and PAC1-Pep3 to preserve fish mobility and tyrosine hydroxylase expression in the brain.ConclusionWe have developed the first neuroprotective pepducins derived from PAC1, a class B GPCR.General significancePAC1-derived pepducins represent attractive templates for the development of innovative neuroprotecting molecules.     

Inhibition of expression of the type I G protein-coupled receptor for vasoactive intestinal peptide (VPAC1) by hammerhead ribozymes

Vasoactive intestinal peptide (VIP) is a neuromediator expressed widely in the nervous, gastrointestinal, respiratory, and immune systems. Two G protein-coupled receptors (GPCRs), designated VPAC1 and VPAC2, bind VIP with high affinity and transduce increases in [cyclic AMP](i) and [Ca(2+)](i). As there are no potent VPAC1- or VPAC2-selective antagonists, a hammerhead ribozyme (Rz) strategy capable of in vivo application was adopted to inactivate individual domains of VPAC1. Three Rzs were designed to cleave mRNA encoding the amino terminus, the third intracellular loop, and the cytoplasmic tail of human VPAC1 and were introduced by transfection into HEK-293 cells expressing recombinant human VPAC1. Each Rz specifically degraded VPAC1 mRNA and down-regulated VPAC1 protein and VIP-binding activity, as assessed by ribonuclease protection assays, Western blots, and binding of (125)I-VIP. Rz-mediated down-regulation of VPAC1 was associated with up to 75% suppression of VIP signaling of increases in [cyclic AMP](i) and [IP3](i), and of cyclic AMP response element-luciferase reports. The Rz specific for the amino terminus inhibited VPAC1 expression and signaling to the greatest extent. VIP-evoked cellular responses thus appear to be proportional to the level of VPAC1 expression. Specific Rzs may be powerful tools for manipulating tissue-specific contributions of GPCRs in vitro and in vivo.     

Multiple G proteins compete for binding with the human gonadotropin releasing hormone receptor

The GnRH receptor is coupled to G proteins of the families G_q and G₁₁. G_q and G₁₁ coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families G_s, G_i, G_q and G₁₁ complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-transfection of HEK293 cells with human WT GnRHR and with stimulatory and inhibitory G proteins (G_q, G₁₁ and G_s, G_i) led to either production or inhibition of total inositol phosphate (IP) production, depending on the G protein that was over-expressed. Studies were conducted in different human (COS7, HeLa) and rodent-derived (CHO-K1, GH₃) cell lines in order to confirm that G protein promiscuity observed with the GnRHR was not limited to a particular cell type.     

Suppression of adenylyl cyclase-mediated cAMP production by plasma membrane associated cytoskeletal protein 4.1G

It has been shown lately that activity of G protein-coupled receptors (GPCRs) is regulated by an array of proteins binding to carboxy (C)-terminus of GPCRs. Proteins of 4.1 family are subsets of subcortical cytoskeletal proteins and are known to stabilize cellular structures and proteins at the plasma membrane. One of the 4.1 family proteins, 4.1G has been shown to interact with the C-terminus of GPCRs and regulate intracellular distribution of the receptors, including parathyroid hormone (PTH)/PTH-related protein receptor (PTHR). PTHR is coupled to trimeric G proteins G_s and G_q, which activate the adenylyl cyclase/cyclic AMP (cAMP) pathway and phospholipase C pathway, respectively. During the course of investigation of the role of 4.1G on adenylyl cyclase/cAMP signaling pathway, we found that 4.1G suppressed forskolin-induced cAMP production in cells. The cAMP accumulation induced by forskolin was decreased in HEK293 cells overexpressing 4.1G or increased in 4.1G-knockdown cells. Furthermore, PTH -(1-34)-stimulated cAMP production was also suppressed in the presence of exogenously expressed 4.1G despite its activity to increase the distribution of PTHR to the cell surface. In cells overexpressing FERM domain-deleted 4.1G, a mutant form of the protein deficient in plasma membrane distribution, neither forskolin-induced nor PTH -(1-34)-stimulated cAMP production was not altered. The suppression of the forskolin-induced cAMP production was observed even in membrane preparations of 4.1G-overexpressing cells. In 4.1G-knockdown HEK293 cells, plasma membrane distribution of adenylyl cyclase 6, one of the major subtypes of the enzyme in the cells, showed a slight decrease, in spite of the increased production of cAMP in those cells when stimulated by forskolin. Also, cytochalasin D treatment did not cause any influence on forskolin-induced cAMP production in HEK293 cells. These data indicate that plasma membrane-associated 4.1G regulates GPCR-mediated G_s signaling by suppressing adenylyl cyclase-mediated cAMP production.     

Gαq-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G_q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G_q family. Classical G_q signalling pathways, or phosphorylation at Ser²²⁵, phospholipase D and Ca²⁺/calmodulin were not involved in M₃ receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G_q-coupled receptors, linking them via cross-activation of S1P receptors to G_i and G_12/13 signalling pathways.     

Specific interaction between the hop1 intracellular loop 3 domain of the human PAC(1) receptor and ARF

The PAC(1), VPAC(1) and VPAC(2) receptors are members of the secretin (Group II) family of G protein-coupled receptors. All members of this family activate adenylate cyclase and several have also been shown to activate phospholipase C. We have recently reported that the rat VPAC(1), VPAC(2) and PAC(1) receptors activate phospholipase D and that distinct pathways are utilised by two intracellular loop 3 splice variants of PAC(1), one of which is ARF-dependent. Phospholipase D activation by the hop1, but not the null (short), form of the PAC(1) receptor is sensitive to brefeldin A, an inhibitor of GTP exchange at ARF. We have expressed the null and hop1 intracellular loop 3 domains of the human PAC(1) receptor in bacteria as GST-fusion proteins and used them as peptide affinity matrices to determine whether a functional interaction exists between these domains and ARF. Using this GST pull-down assay, we have shown binding of the small G protein ARF6 to the hop1 but not the null domain of this receptor.     

Prokineticin receptors interact unselectively with several G protein subtypes but bind selectively to β-arrestin 2

Prokineticin 1 (pk1) and prokineticin 2 (pk2) interact with two structurally related G-protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2). Cellular signalling studies show that the activated receptors can evoke Ca²⁺-mobilization, pertussis toxin-sensitive ERK phosphorylation, and intracellular cAMP accumulation, which suggests the partecipation of several G protein subtypes, such as G_q/11, G_i/o and G_s. However, direct interactions with these transduction proteins have not been studied yet. Here we measured by bioluminescence resonance energy transfer (BRET) the association of PKR1 and PKR2 with different heterotrimeric Gα proteins in response to pk1 and pk2 activation. Using host-cell lines carrying gene deletions of Gα_q/11 or Gα_s, and pertussis toxin treatment to abolish the receptor interactions with Gα_i/o, we determined that both receptors could couple with comparable efficiency to G_q/11 and G_i/o, but far less efficiently to G_s or other pertussis toxin-insensitive G proteins. We also used BRET methodology to assess the association of prokineticin receptors with β-arrestin isoforms. Fluorescent versions of the isoforms were transfected both in HEK293 cells and in double KO β-arrestin 1/2 mouse fibroblasts, to study receptor interaction with the reconstituted individual β-arrestins without background expression of the endogenous genes. Both receptors formed stable BRET-emitting complexes with β-arrestin 2 but not with β-arrestin 1, indicating strong selectivity for the former. In all the studied transducer interactions and in both receptors, pk2 was more potent than pk1 in promoting receptor binding to transduction proteins.     

Synthesis and biological evaluation of Schizandrin derivatives as tubulin polymerization inhibitors

A series of oxime ester-derivatives were prepared by utilizing the schizandrin (1), a major compound isolated from Schisandra grandiflora, which is deployed in different traditional system of medicine. The in vitro antiproliferative activities of the synthesized compounds were assessed against a selected panel of human cancer cell lines (A549, RKO P3, DU145 and Hela) and normal cell (HEK293). Several of these derivatives were found more potent in comparison to parent compound, schizandrin (1). Particularly, 4a and 4b demonstrated potent activity against DU-145 and RKOP3 cell lines with IC₅₀ values of 3.42 µM and 3.35 µM respectively. To characterize the molecular mechanisms involved in antitumoral activity, these two compounds, 4a and 4b were selected for further studies. Cell cycle analysis revealed that both the compounds were able to induce apoptosis and cell cycle arrest at G₀/G₁ phase. To know the extent of apoptosis in DU145 and RKOP3 cell lines, Annexin V-FITC were performed. Moreover, the tubulin polymerization assay indicated that 4a and 4b exhibits potent inhibitory effect on the tubulin assembly. Molecular docking studies and competitive binding assay also indicated that 4a and 4b effectively bind at the colchicine binding site of the tubulin.     

Lysophospholipids and their receptors in the central nervous system

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), two of the best-studied lysophospholipids, are known to influence diverse biological events, including organismal development as well as function and pathogenesis within multiple organ systems. These functional roles are due to a family of at least 11 G protein-coupled receptors (GPCRs), named LPA_1–6 and S1P_1–5, which are widely distributed throughout the body and that activate multiple effector pathways initiated by a range of heterotrimeric G proteins including G_i/o, G_12/13, G_q and G_s, with actual activation dependent on receptor subtypes. In the central nervous system (CNS), a major locus for these signaling pathways, LPA and S1P have been shown to influence myriad responses in neurons and glial cell types through their cognate receptors. These receptor-mediated activities can contribute to disease pathogenesis and have therapeutic relevance to human CNS disorders as demonstrated for multiple sclerosis (MS) and possibly others that include congenital hydrocephalus, ischemic stroke, neurotrauma, neuropsychiatric disorders, developmental disorders, seizures, hearing loss, and Sandhoff disease, based upon the experimental literature. In particular, FTY720 (fingolimod, Gilenya, Novartis Pharma, AG) that becomes an analog of S1P upon phosphorylation, was approved by the FDA in 2010 as a first oral treatment for MS, validating this class of receptors as medicinal targets. This review will provide an overview and update on the biological functions of LPA and S1P signaling in the CNS, with a focus on results from studies using genetic null mutants for LPA and S1P receptors. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

多巴胺受体的cDNA克隆

通过多巴胺受体的5个cDNA克隆,综述和分析了5个多巴胺受体(D₁R-D₅R)的基因结构,在染色体上的定位及其mRNA在中枢脑区的分布;比较了这5个受体cDNA克隆的结构特征和药理学性质.     

VPAC1 (vasoactive intestinal peptide (VIP) receptor type 1) G protein-coupled receptor mediation of VIP enhancement of murine experimental colitis

Distinct roles of the two T cell G protein-coupled receptors for vasoactive intestinal peptide (VIP), termed VPAC1 and VPAC2, in VIP regulation of autoimmune diseases were investigated in the dextran sodium sulfate (DSS)-induced murine acute colitis model for human inflammatory bowel diseases. In mice lacking VPAC2 (VPAC2-KO), DSS-induced colitis appeared more rapidly with greater weight loss and severe histopathology than in wild-type mice. In contrast, DSS-induced colitis in VPAC1-KO mice was milder than in wild-type mice and VPAC2-KO mice. Tissues affected by colitis showed significantly higher levels of myeloperoxidase, IL-6, IL-1β and MMP-9 in VPAC2-KO mice than wild-type mice, but there were no differences for IL-17, IFN-γ, IL-4, or CCR6. Suppression of VPAC1 signals in VPAC2-KO mice by PKA inhibitors reduced the clinical and histological severity of DSS-induced colitis, as well as tissue levels of IL-6, IL-1β and MMP-9. Thus VIP enhancement of the severity of DSS-induced colitis is mediated solely by VPAC1 receptors.     

The amidated PACAP1–23 fragment is a potent reduced-size neuroprotective agent

BackgroundNeurodegenerative disorders, such as Parkinson's disease (PD), are characterized by neuronal death involving, among other events, mitochondrial dysfunction and excitotoxicity. Along these lines, several attempts have been made to slow this pathology but none have been yet discovered. Based on its capacity to cross the blood-brain barrier and provide neuronal protection in vitro and in vivo, the pituitary adenylate cyclase-activating polypeptide (PACAP) represents a promising lead molecule. Pharmacological studies showed that PACAP interacts with three different G protein-coupled receptors, i.e. PAC1, VPAC1 and VPAC2. However, only PAC1 is associated with neuronal anti-apoptotic actions, whilst VPAC activation might cause adverse effects. In the context of the development of PAC1-selective agonists, PACAP(1−23) (PACAP23) appears as the shortest known PACAP bioactive fragment.MethodsHence, the capacity of this peptide to bind PACAP receptors and protect neuroblastoma cells was evaluated under conditions of mitochondrial dysfunction and glutamate excitotoxicity. In addition, its ability to activate downstream signaling events involving G proteins (Gα_s and Gα_q), EPAC, and calcium was also assessed.ResultsCompared to the endogenous peptide, PACAP23 showed a reduced affinity towards PAC1, although this fragment exerted potent neuroprotection. However, surprisingly, some disparities were observed for PACAP23 signaling compared to full length PACAP, suggesting that downstream signaling related to neuroprotection is distinctly regulated following subtle differences in their PAC1 interactions.ConclusionsAltogether, this study demonstrates the potent neuroprotective action of amidated PACAP23.General significancePACAP23 represents an attractive template for development of shorter PACAP-derived neuroprotective molecules.     

A Cleavable N-Terminal Signal Peptide Promotes Widespread Olfactory Receptor Surface Expression in HEK293T Cells

Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and G_αolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and G_αolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies.     

Refolding of G protein alpha subunits from inclusion bodies expressed in Escherichia coli

Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The α subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the G_α family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only 10% of total G_α protein expressed is active; the rest accumulates in inclusion bodies. Although G_iα has been readily expressed in soluble form (to 10 mg/L), other α subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold G_α from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of G_α subunits (human G_iα(1), human G_sα(short), human G_11α and human G_tα(cone)), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble G_iα expressed from E. coli showed that although the refolded G_α subunits were soluble and retained partial α-helicity characteristic of the native, folded G_α subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded G_iα protein has a native-like secondary structure, but is predominately in a molten globular state.     

Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β₂-adrenoceptor (β₂-AR) couples dually to G_s and G_i proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β₂-AR causes a switch in receptor coupling from G_s to G_i. More recent studies have demonstrated that phosphorylation of β₂-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the G_i coupling. We have previously shown that although most β₂-AR agonists cause both G_s and G_i activation, (R,R′)-fenoterol preferentially activates β₂-AR-G_s signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β₂-AR essential for G_s-biased signaling. Following stimulation with a β₂-AR-G_s-biased agonist (R,R′)-4′-aminofenoterol, the G_i disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β₂-AR. Interestingly, Y308F substitution on β₂-AR enabled (R,R′)-4′-aminofenoterol to activate G_i and to produce these responses in a pertussis toxin-sensitive manner without altering β₂-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β₂-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β₂-AR stabilize receptor conformations favoring the receptor-G_s protein coupling and subsequently result in G_s-biased agonism.     

Human melanopsin forms a pigment maximally sensitive to blue light (λmax ≈ 479 nm) supporting activation of Gq/11 and Gi/o signalling cascades

A subset of mammalian retinal ganglion cells expresses an opsin photopigment (melanopsin, Opn4) and is intrinsically photosensitive. The human retina contains melanopsin, but the literature lacks a direct investigation of its spectral sensitivity or G-protein selectivity. Here, we address this deficit by studying physiological responses driven by human melanopsin under heterologous expression in HEK293 cells. Luminescent reporters for common second messenger systems revealed that light induces a high amplitude increase in intracellular calcium and a modest reduction in cAMP in cells expressing human melanopsin, implying that this pigment is able to drive responses via both G_q and G_i/o class G-proteins. Melanopsins from mouse and amphioxus had a similar profile of G-protein coupling in HEK293 cells, but chicken Opn4m and Opn4x pigments exhibited some G_s activity in addition to a strong G_q/11 response. An action spectrum for the calcium response in cells expressing human melanopsin had the predicted form for an opsin : vitamin A1 pigment and peaked at 479 nm. The G-protein selectivity and spectral sensitivity of human melanopsin is similar to that previously described for rodents, supporting the utility of such laboratory animals for developing methods of manipulating this system using light or pharmacological agents.