Spare Receptors Research Articles

PTH1R Actions on Bone Using the cAMP/Protein Kinase A Pathway.

After the initial signaling action of parathyroid hormone (PTH) on bone was shown to be activation of adenylyl cyclase, its target was found to be cells of the osteoblast lineage, to the exclusion of osteoclasts and their precursors. This led to the view that the osteoblast lineage regulated osteoclast formation, a proposal that was established when the molecular mechanisms of osteoclast formation were discovered. This is in addition to the effect of PTH1Rv signaling throughout the osteoblast differentiation process to favour the formation of bone-forming osteoblasts. Initial signaling in the PTH target cells through cAMP and protein kinase A (PKA) activation is extremely rapid, and marked by an amplification process in which the later event, PKA activation, precedes cAMP accumulation in time and is achieved at lower concentrations. All of this is consistent with the existence of “spare receptors”, as is the case with several other peptide hormones. PTH-related protein (PTHrP), that was discovered as a cancer product, shares structural similarity with PTH in the amino-terminal domain that allows the hormone, PTH, and the autocrine/paracrine agent, PTHrP, to share actions upon a common G protein coupled receptor, PTH1R, through which they activate adenylyl cyclase with equivalent potencies. Studies of ligand-receptor kinetics have revealed that the PTH/PTH1R ligand-receptor complex, after initial binding and adenylyl cyclase activation at the plasma membrane, is translocated to the endosome, where adenylyl cyclase activation persists for a further short period. This behavior of the PTH1R resembles that of a number of hormones and other agonists that undergo such endosomal translocation. It remains to be determined whether and to what extent the cellular effects through the PTH1R might be influenced when endosomal is added to plasma membrane activation.

Adenosine Receptor Reserve and Long-Term Potentiation: Unconventional Adaptive Mechanisms in Cardiovascular Diseases?

While the concept of a receptor reserve (spare receptors) is old, their presence on human cells as an adaptive mechanism in cardiovascular disease is a new suggestion. The presence of spare receptors is suspected when the activation of a weak fraction of receptors leads to maximal biological effects, in other words, when the half-maximal effective concentration (EC50) for a biological effect (cAMP production, for example) is lower than the affinity (KD) of the ligand for a receptor. Adenosine is an ATP derivative that strongly impacts the cardiovascular system via its four membrane receptors, named A1R, A2AR, A2BR, and A3R, with the A1R being more particularly involved in heart rhythm, while the A2AR controls vasodilation. After a general description of the tools necessary to explore the presence of spare receptors, this review focuses on the consequences of the presence of spare adenosine receptors in cardiovascular physiopathology. Finally, the role of the adenosinergic system in the long-term potentiation and its possible consequences on the physiopathology are also mentioned.

Roles of hepatic atypical protein kinase C hyperactivity and hyperinsulinemia in insulin-resistant forms of obesity and type 2 diabetes mellitus.

Diet‐induced obesity, the metabolic syndrome, type 2 diabetes (DIO/MetS/T2DM), and their adverse sequelae have reached pandemic levels. In mice, DIO/MetS/T2DM initiation involves diet‐dependent increases in lipids that activate hepatic atypical PKC (aPKC) and thereby increase lipogenic enzymes and proinflammatory cytokines. These or other hepatic aberrations, via adverse liver‐to‐muscle cross talk, rapidly impair postreceptor insulin signaling to glucose transport in muscle. The ensuing hyperinsulinemia further activates hepatic aPKC, which first blocks the ability of Akt to suppress gluconeogenic enzyme expression, and later impairs Akt activation, further increasing hepatic glucose production. Recent findings suggest that hepatic aPKC also increases a proteolytic enzyme that degrades insulin receptors. Fortunately, all hepatic aberrations and muscle impairments are prevented/reversed by inhibition or deficiency of hepatic aPKC. But, in the absence of treatment, hyperinsulinemia induces adverse events, some by using “spare receptors” to bypass receptor defects. Thus, in brain, hyperinsulinemia increases Aβ‐plaque precursors and Alzheimer risk; in kidney, hyperinsulinemia activates the renin–angiotensin–adrenal axis, thus increasing vasoconstriction, sodium retention, and cardiovascular risk; and in liver, hyperinsulinemia increases lipogenesis, obesity, hepatosteatosis, hyperlipidemia, and cardiovascular risk. In summary, increases in hepatic aPKC are critically required for development of DIO/MetS/T2DM and its adverse sequelae, and therapeutic approaches that limit hepatic aPKC may be particularly effective.

Recent advances in the role of the adenosinergic system in coronary artery disease.

Adenosine is an endogenous nucleoside that plays a major role in the physiology and physiopathology of the coronary artery system, mainly by activating its A2A receptors (A2AR). Adenosine is released by myocardial, endothelial, and immune cells during hypoxia, ischaemia, or inflammation, each condition being present in coronary artery disease (CAD). While activation of A2AR improves coronary blood circulation and leads to anti-inflammatory effects, down-regulation of A2AR has many deleterious effects during CAD. A decrease in the level and/or activity of A2AR leads to: (i) lack of vasodilation, which decreases blood flow, leading to a decrease in myocardial oxygenation and tissue hypoxia; (ii) an increase in the immune response, favouring inflammation; and (iii) platelet aggregation, which therefore participates, in part, in the formation of a fibrin-platelet thrombus after the rupture or erosion of the plaque, leading to the occurrence of acute coronary syndrome. Inflammation contributes to the development of atherosclerosis, leading to myocardial ischaemia, which in turn leads to tissue hypoxia. Therefore, a vicious circle is created that maintains and aggravates CAD. In some cases, studying the adenosinergic profile can help assess the severity of CAD. In fact, inducible ischaemia in CAD patients, as assessed by exercise stress test or fractional flow reserve, is associated with the presence of a reserve of A2AR called spare receptors. The purpose of this review is to present emerging experimental evidence supporting the existence of this adaptive adenosinergic response to ischaemia or inflammation in CAD. We believe that we have achieved a breakthrough in the understanding and modelling of spare A2AR, based upon a new concept allowing for a new and non-invasive CAD management.

Adenosine Receptor Profiling Reveals an Association between the Presence of Spare Receptors and Cardiovascular Disorders.

Adenosine and its receptors exert a potent control on the cardiovascular system. This review aims to present emerging experimental evidence supporting the existence and implication in cardiovascular disorders of specific adenosinergic pharmacological profiles, conforming to the concept of “receptor reserve”, also known as “spare receptors”. This kind of receptors allow agonists to achieve their maximal effect without occupying all of the relevant cell receptors. In the cardiovascular system, spare adenosine receptors appear to compensate for a low extracellular adenosine level and/or a low adenosine receptor number, such as in coronary artery disease or some kinds of neurocardiogenic syncopes. In both cases, the presence of spare receptors appears to be an attempt to overcome a weak interaction between adenosine and its receptors. The identification of adenosine spare receptors in cardiovascular disorders may be helpful for diagnostic purposes.

Pharmacological profile of adenosine A2A receptors in patients with lower extremity peripheral artery disease and associated coronary artery disease: A pilot study

BackgroundAltered blood flow occurs in patients with low extremity peripheral artery disease (LE-PAD). LE-PAD is mostly associated with coronary artery disease (CAD). Adenosine is an endogenous nucleoside that affects both coronary and limb artery blood flow, mostly via the adenosine A2A receptor (A2AR). We evaluated A2AR expression and function in peripheral blood mononuclear cells (PBMCs) and the femoral artery tissues of patients with LE-PAD. MethodsArtery tissues and PBMCs were sampled in 24 patients with intermittent claudication, and compared with PBMCs in 24 healthy subjects. Expression and function of A2AR was studied, using a A2AR monoclonal antibody with agonist properties, allowing determination of A2AR affinity (KD) and cAMP production (ie.EC50). ResultsA2AR expression on PBMCs was lower in patients than controls (median1.3 [range 0.6–1.8] vs 1.75 [1.45–2.1] arbitrary units; P < 0.01), and correlated with A2AR expression in artery tissues (Pearson's r = 0.71; P < 0.01). Basal and maximally stimulated cAMP production of PBMCs was lower in patients vs controls: 172 [90–310] vs 244 [110–380] pg/106 cells (P < 0.05) and 375 [160–659] vs 670 [410–980] pg/106 cells (P < 0.05), respectively. A high KD/EC50 ratio, characteristic of spare receptors, was observed in CAD with inducible-myocardial-ischemia. ConclusionA2AR expression in the arteries of patients, correlated with their expression in PBMCs. A2AR expression was lower in patients than in controls. A single blood sample (for measurement of A2AR expression on PBMCs) may help to screen patients with LE-PAD, whereas the presence of spare receptors may help with risk stratification before vascular surgery in CAD patients with high risk of myocardial ischemia.

Kinetics of oxytocin and deaminooxytocin displacement from the OXTR-receptor compartment in rat uterus ex vivo

The oil immersion method suggested earlier by Kalsner and Nickerson for analysing actions of sympathomimetic drugs upon smooth muscle tissues was applied to isometric preparations of rat myometrium stimulated by oxytocin and deaminooxytocin. An exchange of the aqueous medium by mineral oil allows monitoring the displacement of the peptides from their receptor compartment in absence of free diffusion transport between tissue and organ medium. Exponential analysis of the data from the uterotonic decay phase allows several inferences to be drawn: 1) Transport rate constants (roughly equal for the two peptides) are higher than rate constants of (irreversible) elimination from the receptor compartment. 2) The response decay rate in the oil immersion phase is proportional solely to the peptide elimination and thus offers estimates of elimination rate constants. 3) Peptide elimination kinetics in the receptor compartment is only insignificantly influenced by the kinetics of ligand-receptor binding. 4) As expected, the elimination rate constant of deaminooxytocin is considerably lower than for oxytocin. The apparent concentration of receptors in the paracellular space of the myometrium (“apparent”, since receptor molecules are embedded in the cell membrane and hence not exposed to a diffusive flux), estimated from histometric parameters, appears rather high: 7 and 120 μM for high and low affinity receptors, respectively. Concentration-response curves for rat uterus stimulated by oxytocin or deaminooxytocin indicate that only about 0.25 to 5 per cent of the available receptors are involved in eliciting a maximal uterus contraction. The remnant receptor pool is likely to behave as a receptor reserve (“spare receptors”).

Spare Adenosine A2a Receptors Are Associated With Positive Exercise Stress Test In Coronary Artery Disease.

During exercise, cardiac oxygen-consumption increases and the resulting low oxygen level in myocardium triggers coronary vasodilation. This response to hypoxia is controlled notably by the vasodilator adenosine and its A2A receptor (A2AR). According to the "spare receptor" pharmacological model, a strong A2AR-mediated response can occur in the context of a large number of receptors remaining unoccupied, activation of only a weak fraction of A2AR (evaluated using KD) resulting in maximal cAMP production (evaluated using EC50), and hence in maximal coronary vasodilation. In coronary artery disease (CAD), myocardial ischemia limits adaptation to exercise, which is commonly detected using the exercise stress test (EST). We hypothesized that spare A2AR are present in CAD patients to correct ischemia. Seventeen patients with angiographically-documented CAD and 17 control subjects were studied. We addressed adenosine-plasma concentration and A2AR-expression at the mononuclear cell-surface, which reflects cardiovascular expression. The presence of spare A2AR was tested using an innovative pharmacological approach based on a homemade monoclonal antibody with agonist properties. EST was positive in 82% of patients, and in none of the controls. Adenosine plasma-concentration increased by 60% at peak exercise in patients only (p<0.01). Most patients (65%), and none of the controls, had spare A2AR (identified when EC50/KD≤0.1) and a low A2AR-expression (mean: -37% vs controls; p<0.01). All patients with spare A2AR had a positive EST whereas the subjects without spare A2AR had a negative EST (p<0.05). Spare A2AR are therefore associated with positive EST in CAD patients and their detection may be used as a diagnostic marker.

Superagonism at G protein‐coupled receptors and beyond

Ligands targeting GPCRs can be categorized according to their intrinsic efficacy to trigger a specific, receptor-mediated response. A ligand endowed with the same level of efficacy as the endogenous agonist can be classified as a full agonist, whereas a compound that displays greater efficacy, that is, higher receptor signalling output than the endogenous agonist, can be called a superagonist. Subsequent to GPCR activation, an intracellular signalling cascade is set in motion, which may generate substantial amplification of the signal. This may obscure superagonism in pharmacological assays and, therefore, the definition of superagonism necessitates a combination of operational approaches, reduction of spare receptors or estimation of receptor activation close to the receptor level to quantify relative agonist efficacies in a particular system. The first part of this review will compare GPCR superagonism with superagonism in the field of immunology, where this term is well established. In the second part, known GPCR superagonists will be reviewed. Then, the experimental and analytical challenges in the deconvolution of GPCR superagonism will be addressed. Finally, the potential benefit of superagonism is discussed. The molecular mechanisms behind GPCR superagonism are not completely understood. However, crystallography shows that agonist binding alone is not sufficient for a fully active receptor state and that binding of the G protein is at least equally important. Accordingly, the emerging number of reported superagonists implies that ligand-induced receptor conformations more active than the ones stabilized by the endogenous agonist are indeed feasible. Superagonists may have therapeutic potential when receptor function is impaired or to induce negative feedback mechanisms. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Assessing Gonadotropin Receptor Function by Resonance Energy Transfer-Based Assays.

Gonadotropin receptors belong to the super family of G protein-coupled receptors and mediate the physiological effects of follicle-stimulating hormone (FSHR) and luteinizing hormone (LHR). Their central role in the control of reproductive function has made them the focus of intensive studies. Upon binding to their cognate hormone, they trigger complex signaling and trafficking mechanisms that are tightly regulated in concentration, time, and space. Classical cellular assays often fail to capture all these dynamics. Here, we describe the use of various bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays to investigate the activation and regulation of FSHR and LHR in real-time, in living cells (i.e., transiently expressed in human embryonic kidney 293 cells). Indeed, the dynamics of hormone-mediated heterotrimeric G protein activation, cyclic adenosine-monophosphate (cAMP) production, calcium release, β-arrestin 2 recruitment, and receptor internalization/recycling was assessed. Kinetics and dose–response analyses confirmed the expected pharmacological and signaling properties of hFSHR and hLHR but revealed interesting characteristics when considering the two major pathways (cAMP and β-arrestin 2) of the two receptors assessed by BRET. Indeed, the EC50 values were in picomolar range for cAMP production while nanomolar range was observed for β-arrestin 2 recruitment as well as receptor internalization. Interestingly, the predicted receptor occupancy indicates that the maximal G protein activation and cAMP response occur at <10% of receptor occupancy whereas >90% of activated receptors is required to achieve full β-arrestin 2 recruitment and subsequent receptor internalization. The rapid receptor internalization was also followed by a recycling phase. Collectively, our data reveal that β-arrestin-mediated desensitization, internalization, and the subsequent fast recycling of receptors at the plasma membrane may provide a mechanistic ground to the “spare receptor” paradigm. More generally, the novel tools described here will undoubtedly provide the scientific community investigating gonadotropin receptors with powerful means to decipher their pharmacology and signaling with the prospect of pathophysiological and drug discovery applications.

Mu opioid receptors hyperpolarize respiratory‐controlling Kölliker‐Fuse neurons (712.1)

Respiratory depression is the primary cause of death from opioid overdose, yet little is known about the cellular mechanisms of opioids on respiratory‐controlling neurons. The pontine Kölliker‐Fuse (KF) regulates respiration, especially the inspiratory/expiratory phase transition. In the in situ arterially perfused working heart‐brainstem preparation of rat, injection of the mu opioid agonist DAMGO into the KF caused robust apneusis that was similar to complete silencing of the KF. The apneusis was rapidly reversed by perfusion of the opioid antagonist naloxone. In whole‐cell recordings from KF neurons contained in rat brain slices, activation of mu opioid receptors hyperpolarized a majority (75%) of KF neurons. The hyperpolarizing current produced by [Met5]enkephalin (ME) was concentration‐dependent, reversed at the potassium equilibrium potential and was blocked by BaCl2, characteristics of a GIRK conductance. Unexpectedly, the partial agonist morphine produced the same amplitude current as full agonists DAMGO or ME, indicating a large receptor reserve. The presence of many spare receptors could reduce the degree of desensitization induced by prolonged application of ME. Indeed, only minimal acute desensitization of the ME‐mediated GIRK current was observed. This lack of desensitization correlates with the lack of tolerance to the respiratory depressant effects of opioids.Grant Funding Source: Supported by DA08163 (JTW) and DA33036 (ESL)

Irreversible antagonism of receptors accelerates rat cocaine self‐administration behavior (848.4)

A pharmacological model of self‐administration can be explained by the equation T=ln(1+Du/Dst)/k, where T=time between self‐administrations, Du=unit dose, Dst=satiety threshold, and k=first order elimination rate constant, predicts that increasing the pharmacodynamic parameter Dst accelerates self‐administration. Competitive dopamine receptor antagonists temporarily increase cocaine intake implicating dopamine receptor fractional occupancy by dopamine as a mediator of the behavior. Competitive antagonists do not alter the number of receptors, so they cannot be used to study spare receptors or to differentiate between fractional occupancy of receptors and absolute numbers of receptors occupied. Rats that reliably self‐administered cocaine were injected with the non‐specific irreversible antagonist, N‐Ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (1mg/kg i.v.). Attempts were made every 8 hours to reinstate self‐administration and once reinstated self‐administration was regular but was initially 2‐fold higher than baseline with a concomitant increase in the satiety threshold. Over 6 days the rate slowly declined and eventually returned to baseline presumably due to receptor repopulation. This indicates that satiety threshold corresponds to a specific number of receptors occupied. Changes in the number of dopamine receptors are key regulators of addictive behavior.Grant Funding Source: NIH Grant DP1DA031386

NF-κB Activation Mediates Induction Of Anti-Inflammatory Adenosine A2A Receptors In iNKT Cells Of Sickle Cell Patients During Vaso-Occlusive Episodes and Upon Activation Of Cultured Human iNKT Cells

iNKT cells are activated in mice and people with vaso-occlusive sickle cell disease (SCD) and play a pivotal role in initiating an inflammatory cascade. In mice, iNKT cell activation requires NF-κB signaling [Stanic, AK et al. J Immunol. 172:4667, 2004]. In mice with SCD, lung inflammation is associated with induction in iNKT cells of IFN-γ and anti-inflammatory adenosine A2A receptor (A2AR) mRNAs [Wallace, KL et al. Blood 116:5010, 2010]. The A2AR is one of four G protein coupled adenosine receptors (A1, A2A, A2B and A3) and it is the most abundant adenosine receptor expressed in T cells. In patients with SCD, painful vaso-occlusive crisis (pVOC) is associated with NF-κB activation and increased expression of A2AR immunoreactivity detected with the monoclonal Ab, 7F6-G5-A2, in circulating iNKT cells. Infusion of a moderate dose (6 nM) of the A2AR agonist, regadenoson during pVOC reduced iNKT cell activation over time [Field, J. et al. Blood 121:3329, 2013]. Since A2AR activation inhibits iNKT cell activation, we reasoned that iNKT cell activation during pVOC might occur preferentially in iNKT cells with low A2AR expression. However, in the current study we show that activated human Vα24+ CD4+ iNKT cells concordantly (i.e. on the same cells) expressed Ser-536 phosphorylated/activated NF-κB (p-NFκB), cytokines and A2ARs. These findings suggest that NF-κB signaling increases A2AR expression as a counter-regulatory mechanism to render activated iNKT cells highly sensitive to inhibition by A2AR activation. Spare receptor theory predicts that enhanced receptor expression will increase the functional potency of agonists. To test this hypothesis we infused into stable SCD patients at steady state a low dose of regadenoson (2 nM) well below its receptor binding KD that was measured by competition for radioligand binding to be 12 nM. Low-dose regadenoson infusion reduced the percentage of p-NF-κB-high/activated iNKT cells by an average of 69% over 12 hours (P = 0.005). These findings suggest that A2AR induction is a counter-regulatory mechanism that limits iNKT cell activation by increasing the sensitivity of activated iNKT cells to inhibition by adenosine. In order to further test the hypothesis that NF-κB activation stimulates A2AR transcription, we examined cultured human iNKT cells. Blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of cytokine and A2AR mRNA and protein upon iNKT activation by plate-bound anti-CD3 antibodies. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells serves as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. We conclude that A2AR agonists have therapeutic potential for treating inflammatory diseases associated with iNKT cell activation, including SCD. Disclosures:Off Label Use: Ragadenoson is used to inhibit activation of iNKT cells.

Search for adenosine A2A spare receptors on peripheral human lymphocytes

Some ligand–receptor couples involve spare receptors, which are apparent when a maximal response is achieved with only a small fraction of the receptor population occupied. This situation favours cross-reactions with low-affinity ligands, which may be detrimental for cell signaling. In the case of the adenosine A2A receptors (A2AR), which have an immunosuppressive effect on lymphocytes through cAMP production, the presence of spare A2AR remains to be established. We examined the situation using patients over-expressing lymphocyte A2AR and an agonist-like mAb to A2AR. We found that maximal mAb binding and functional response varied among the patients whereas the dissociation constant and half-maximal effective concentration had similar mean values (0.19 and 0.18 μM, respectively). Lymphocyte A2AR expression was correlated to plasma adenosine level and A2AR occupation but not to A2AR response. These results are consistent with a lack of a reserve of functional A2AR on human lymphocytes as a general rule and suggest that the amount and functional state of the expressed A2AR determine the maximal level of the lymphocyte response to adenosine.

Glucagon and Cyclic AMP: Time to Turn the Page?

It is well established that glucagon can stimulate adipose lipolysis, myocardial contractility, and hepatic glucose output by activating a GPCR and adenylate cyclase (AC) and increasing cAMP production. It is also widely reported that activation of AC in all three tissues requires pharmacological levels of the hormone, exceeding 0.1 nM. Extensive evidence is presented here supporting the view that cAMP does not mediate metabolic actions of glucagon on adipose, heart, or liver in vivo. Only pharmacological levels stimulate AC, adipose lipolysis, or cardiac contractility. Physiological concentrations of glucagon (below 0.1 nM) duplicate metabolic effects of insulin on the heart by activating a PI3K-dependent signal without stimulating AC. In the liver, glucagon can enhance gluconeogenesis and glucose output - by increasing the expression of PEPCK or inhibiting the activity of PK - at pharmacological concentrations by activating AC coupled to a low-affinity GPCR, but also at physiological concentrations by activating a high affinity receptor without generating cAMP. Plausible AC/cAMP-independent signals mediating the increase in gluconeogenesis include p38 MAPK (PEPCK expression) and IP3/DAG/Ca(2+) (PK activity). None of glucagon's physiological effects can be explained by activation of spare receptors or amplification of the AC/cAMP signal. In a new model proposed here, glucagon antagonizes insulin on the liver but mimics insulin on the heart without activating AC. Confirmation of the model would have broad implications, applicable not only to the general field of metabolic endocrinology but also to the specific role of glucagon in the pathogenesis and treatment of diabetes.

Specific targeting of pancreatic β‐cells by simultaneous binding of GLP‐1 and α2‐adrenergic receptors with a multivalent ligand

Diabetes Mellitus is characterized by a decline in β‐cell mass and function. The lack of specificity of β‐cell targeting agents poses a challenge to observe changes in β‐cell mass and thus monitor disease progression. We propose β‐cell specificity can be enhanced by using multivalent ligands that simultaneously bind to multiple unique surface receptors that, as a combination, identify a cell type of interest. We synthesized a ligand composed of Yohimbine (Yhb) (α2 adrenergic receptor (α2AR) antagonist) linked to Glucagon‐like Peptide 1(GLP‐1). Receptors to both ligands are expressed on βTC3 cells at a ratio of ~ 1.5:1 (GLP‐1R/α2AR). Microscopy of a fluorophore tagged ligand indicated that binding was rapid with removal of ligand from the cell surface seen within 2–3 min. From population based competition assays, the binding affinity for monovalent GLP‐1 was ~10 nM, whereas GLP‐1/Yhb exhibited 2 distinct binding sites. The high affinity binding (9 pM) is correlated to MVL crosslinking the complementary receptors causing a decrease in ligand off rate, resulting in a 1000X increase in apparent affinity. The low affinity binding (225 nM) is consistent with weak binding of the GLP‐1 moiety in the MVL to the spare GLP‐1 receptors in βTC3 cells. Our findings suggest that GLP‐1/Yhb acts as a specific targeting agent to β‐cells as it exhibits enhanced binding to and retention in β‐cells compared to other cells in the imaging field.Supported by: Juvenile Diabetes Research Foundation, and the Arizona Biomedical Research Commission

Occupancy of both sites on the thyrotropin (TSH) receptor dimer is necessary for phosphoinositide signaling

The thyroid-stimulating hormone (TSH) receptor signals via G(s) to produce cAMP and via G(q/11) to produce inositol-1,4,5-trisphosphate, which is degraded to inositol monophosphate (IP1; phosphoinositide signaling). The potency of TSH for cAMP signaling is higher than for phosphoinositide signaling, and it was suggested that there are "spare receptors" for cAMP signaling. In a human embryonic kidney macrophage scavenger receptor-expressing (HEK-EM) 293 model system, there are no spare receptors, but the cells still exhibited 100-fold differences in potencies. Dose responses for TSH-stimulated dissociation of prebound (125)I-TSH (negative cooperativity; EC(50)=70 mU/ml), which requires TSH binding to both sites of the TSH receptor (TSHR) homodimer, and TSH-stimulated IP1 production (EC(50)=50 mU/ml) were indistinguishable. Fluorescence resonance energy transfer (FRET) using tagged receptors showed that TSHR formed homodimers and heterodimers with two binding-deficient mutant TSHRs, L252P and C41S. When L252P or C41S was expressed with TSHR, that is, when TSHR/L252P or TSHR/C41S heterodimers could only bind one TSH, TSH-stimulated IP1 production was decreased relative to cAMP production. The slopes of linear regression analyses comparing fold stimulation by TSH of IP1 vs. cAMP production were 0.044 ± 0.0047, 0.0043 ± 0.0041, and 0.0059 ± 0.0014 for cells expressing TSHR alone, TSHR and L252P, or TSHR and C41S, respectively. We suggest that TSHR coupling to phosphoinositide signaling is dependent on binding 2 molecules of TSH to TSHR homodimer, causing a conformational change allowing coupling to G(q/11).

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

Adenosine – A Physiological Regulator and a Distress Signal

Abstract: The present brief review argues the case that adenosine can be both a distress signal and a physiological regula-tor. A key factor in determining which of these possibilities pertain is related to the number of receptors expressed. As the signaling from the adenosine receptor to the functional response generally involves amplification, we have a situation in-volving so called spare receptors. This has the consequence that alterations in the receptor number lead to shifts in the po-tency of the endogenous agonist rather than a shift in the maximum response elicited. The roles of adenosine are studied by antagonists and/or animals (mostly mice) with targeted deletions of receptors or enzymes involved in adenosine me-tabolism. Whereas, adaptive changes in the genetically modified mice can occur for the physiologically important effects, such adaptive changes are less likely to occur for the situations when adenosine acts as a distress signal. Keywords: Adenosine receptors, ATP, genetically modified mice, receptor reserve. There are four evolutionarily well conserved receptors for adenosine denoted A

Agonist efficacy and receptor desensitization: from partial truths to a fuller picture

It has been demonstrated that the degree of agonist-induced desensitization of the beta(2)-adrenoceptor is related to agonist efficacy (strength of signalling), whereby high-efficacy agonists (e.g. formoterol) cause more phosphorylation and internalization of the receptor than low-efficacy agonists (e.g. salmeterol). These early studies, however, used a protocol where agonists were matched for receptor occupancy rather than functional effect. In this issue of the BJP, Duringer and colleagues have extended these studies to compare the ability of agonists to cause desensitization at equi-effective (cAMP signalling) concentrations rather than equal occupancy. Their data and conclusions are quite different from those previously described. After prolonged exposure, all the agonists caused a similar degree of desensitization, whereas a pulse protocol uncovered a greater loss of responsiveness with the low-efficacy ligands. This is consistent with the notion that high-efficacy agonists have 'spare receptors', and are therefore less sensitive to loss of receptors through desensitization. It also reflects experience in the clinic, where both formoterol and salmeterol show a similar early decline in bronchoprotection, after which their effects remain stable. These findings challenge the notion that high-efficacy ligands always cause more functional desensitization.