Total Receptor Number Research Articles

Lipid rafts and membrane cholesterol are involved in regulating D2 dopamine receptor signaling

Lipid rafts are specialized membrane microdomains enriched in cholesterol, sphingolipids and caveolin. They are important in the organization of GPCR‐protein complexes and the regulation of signaling. Given the emerging significance of lipids with respect to GPCR structure and activation, we have investigated the role of lipid rafts and membrane cholesterol on D2 dopamine receptor (DAR) localization and function. Sucrose density fractionation revealed that D2 DARs are enriched in lipid rafts from both striatum and transfected HEK293 cells. Depletion of membrane cholesterol with methyl‐β‐cyclodextrin (MCD) diminished cell‐surface D2 DAR expression in HEK293T cells by approximately 75% without altering total receptor number. MCD treatment also completely abolished DA signaling at the D2 DAR. Importantly, when D2 DAR transfections were titrated to achieve surface expression values comparable to MCD‐treatment, a DA‐dependent decrease in cAMP was still observed. Diminished signaling of Gi/o‐coupled muscarinic receptors was also observed following MCD‐treatments. Notably, MCD‐treatment did not inhibit Gs‐linked D1 DAR function or forskolin‐stimulated cAMP accumulation. These results suggest that lipid rafts and/or membrane cholesterol are critical for D2 DAR signaling.

Andrographolide regulates epidermal growth factor receptor and transferrin receptor trafficking in epidermoid carcinoma (A‐431) cells

Andrographolide is the active component of Andrographis paniculata, a plant used in both Indian and Chinese traditional medicine, and it has been demonstrated to induce apoptosis in different cancer cell lines. However, not much is known about how it may affect the key receptors implicated in cancer. Knowledge of how andrographolide affects receptor trafficking will allow us to better understand new mechanisms by which andrographolide may cause death in cancer cells. We utilized the well-characterized epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) expressed in epidermoid carcinoma (A-431) cells as a model to study the effect of andrographolide on receptor trafficking. Receptor distribution, the total number of receptors and surface receptors were analysed by immunofluorescence, Western blot as well as flow-cytometry respectively. Andrographolide treatment inhibited cell growth, down-regulated EGFRs on the cell surface and affected the degradation of EGFRs and TfRs. The EGFR was internalized into the cell at an increased rate, and accumulated in a compartment that co-localizes with the lysosomal-associated membrane protein in the late endosomes. This study sheds light on how andrographolide may affect receptor trafficking by inhibiting receptor movement from the late endosomes to lysosomes. The down-regulation of EGFR from the cell surface also indicates a new mechanism by which andrographolide may induce cancer cell death.

Reduced size of the dendritic tree does not protect Purkinje cells from excitotoxic death

Purkinje cell loss by excitotoxic damage is a typical finding in many cerebellar diseases. One important aspect of this high sensitivity of Purkinje cells to excitotoxic death might be the enormous size of their dendritic tree, with a high load of excitatory glutamate receptors. We have studied whether reduction in the size of the dendritic tree might confer resistance against excitotoxic death to Purkinje cells. We have grown Purkinje cells in organotypic cerebellar slice cultures under chronic activation of metabotropic glutamate receptors or of protein kinase C. Both treatments strongly reduced dendritic tree size. After this treatment, cells were exposed to the glutamate receptor agonist AMPA, which has a strong excitotoxic effect on Purkinje cells. We found that Purkinje cells with small dendritic trees were as sensitive to AMPA exposure as untreated control cells with large dendritic trees. Immunostaining against vesicular glutamate transporter 1 revealed that the small dendritic trees were densely covered by glutamatergic terminals. Our results indicate that the expansion of the dendritic tree and the total number of AMPA receptors per neuron do not play a major role in determining the susceptibility of Purkinje cells to excitotoxic death.

Reduced expression and desensitization of adenosine A 1 receptor/adenylyl cyclase pathway after chronic (−)N 6-phenylisopropyladenosine intake during pregnancy

Little is known about the G protein–coupled receptor desensitization process during pregnancy. Wistar pregnant rats were treated with (−)N 6-phenylisopropyladenosine (R-PIA), an adenosine A 1 receptor (A 1R) agonist, in their drinking water during pregnancy, and the effect on A 1R/adenylyl cyclase system was studied in both maternal and fetal brain. In maternal brain, binding assays revealed a significant decrease in total receptor numbers in plasma membranes (27%, P<0.05), with no significant changes in receptor affinity. The effect of R-PIA on plasma membranes from fetal brains was more marked, with approximately 42% ( P<0.05) of the total receptors detected in control fetuses. Real time reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that chronic R-PIA treatment during the whole gestational period only decreased significantly mRNA level coding A 1R in maternal brain ( P<0.05). αGi 1,2 and αGi 3 subunits were not affected in mothers or fetuses as revealed by immunoblotting. mRNA levels coding these subunits were also unaffected in mothers and fetuses. On the other hand, forskolin- and forskolin-plus guanosine-5′-O-(3-thiotriphosphate) (GTPγS)–stimulated adenylyl cyclase activity was decreased in maternal ( P<.01) and fetal brain ( P<.001). Furthermore, adenylyl cyclase inhibition elicited by N 6-cyclohexyladenosine (CHA), a selective A 1R agonist, was significantly decreased in both maternal ( P<0.05) and fetal brain ( P<.01), suggesting a desensitization of the A 1R/adenylyl cyclase pathway. Therefore, these results suggest that R-PIA intake during pregnancy causes desensitization of the A 1R-mediated inhibitory transduction pathway in both maternal and fetal brain, probably due to the decreased density of A 1R at the cell surface.

Nonlinear pharmacokinetics of therapeutic proteins resulting from receptor mediated endocytosis

Receptor mediated endocytosis (RME) plays a major role in the disposition of therapeutic protein drugs in the body. It is suspected to be a major source of nonlinear pharmacokinetic behavior observed in clinical pharmacokinetic data. So far, mostly empirical or semi-mechanistic approaches have been used to represent RME. A thorough understanding of the impact of the properties of the drug and of the receptor system on the resulting nonlinear disposition is still missing, as is how to best represent RME in pharmacokinetic models. In this article, we present a detailed mechanistic model of RME that explicitly takes into account receptor binding and trafficking inside the cell and that is used to derive reduced models of RME which retain a mechanistic interpretation. We find that RME can be described by an extended Michaelis–Menten model that accounts for both the distribution and the elimination aspect of RME. If the amount of drug in the receptor system is negligible a standard Michaelis–Menten model is capable of describing the elimination by RME. Notably, a receptor system can efficiently eliminate drug from the extracellular space even if the total number of receptors is small. We find that drug elimination by RME can result in substantial nonlinear pharmacokinetics. The extent of nonlinearity is higher for drug/receptor systems with higher receptor availability at the membrane, or faster internalization and degradation of extracellular drug. Our approach is exemplified for the epidermal growth factor receptor system.Electronic supplementary materialThe online version of this article (doi:10.1007/s10928-009-9120-1) contains supplementary material, which is available to authorized users.

Essential Role of Hrs in Endocytic Recycling of Full-length TrkB Receptor but Not Its Isoform TrkB.T1

Brain-derived neurotrophic factor (BDNF) signaling through its receptor, TrkB, modulates survival, differentiation, and synaptic activity of neurons. Both full-length TrkB (TrkB-FL) and its isoform T1 (TrkB.T1) receptors are expressed in neurons; however, whether they follow the same endocytic pathway after BDNF treatment is not known. In this study we report that TrkB-FL and TrkB.T1 receptors traverse divergent endocytic pathways after binding to BDNF. We provide evidence that in neurons TrkB.T1 receptors predominantly recycle back to the cell surface by a "default" mechanism. However, endocytosed TrkB-FL receptors recycle to a lesser extent in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner which relies on its tyrosine kinase activity. The distinct role of Hrs in promoting recycling of internalized TrkB-FL receptors is independent of its ubiquitin-interacting motif. Moreover, Hrs-sensitive TrkB-FL recycling plays a role in BDNF-induced prolonged mitogen-activated protein kinase (MAPK) activation. These observations provide evidence for differential postendocytic sorting of TrkB-FL and TrkB.T1 receptors to alternate intracellular pathways.

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats.

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (Vmax) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (Bmax) and affinity (Kd) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.

Membrane cholesterol removal promotes a low agonist affinity state of mu opioid receptors

Previously, we have shown that removal of plasma membrane cholesterol by the cholesterol‐sequestering agent methyl‐β‐cyclodextrin (MβCD) results in a loss of potency of the mu opioid agonist DAMGO to stimulate GTPγ35S binding or to inhibit adenylyl cyclase. Here, we test the hypothesis that this decrease in potency is due to an uncoupling of the mu opioid receptor from G protein. Approximately half of the mu opioid receptors stably expressed in HEK293 cells were present on the plasma membrane, as determined by whole cell binding. Removal of plasma membrane cholesterol with MβCD significantly decreased the maximum binding of the mu opioid agonist 3H‐DAMGO in a cell membrane preparation. This decrease in agonist binding was partially reversed when cholesterol was added back to the membrane, and could not be explained by changes in total receptor number as measured by binding of the opioid antagonist 3H‐diprenorphine. Rather, MβCD treatment induced the appearance of a low affinity state of the mu opioid receptor as shown by competition of DAMGO with 3H‐diprenorphine. This induced low affinity state was mimicked by the addition of sodium and GTPγS, which uncouples G proteins from receptors. Thus, removal of plasma membrane cholesterol by MβCD decreased the high affinity state of mu opioid receptors which could be explained by an uncoupling of receptor from G protein. Supported by DA07267, DA23339, GM07767, DA04087.

A Dynamic Corral Model of Receptor Trafficking at a Synapse

The postsynaptic density (PSD) is a cytoskeletal specialization within the postsynaptic membrane of a neuron that helps to concentrate and organize neurotransmitter receptors at a chemical synapse. The total number of receptors within the PSD, which is a major factor in determining the physiological strength or weight of a synapse, fluctuates due to the surface diffusion of receptors into and out of the PSD, and the interactions of receptors with scaffolding proteins and cytoskeletal elements within the PSD. In this article, we present a stochastic model of protein receptor trafficking at the PSD that takes into account these various processes. The PSD is treated as a stochastically gated corral, which contributes a source of extrinsic or environmental noise that supplements the intrinsic noise arising from small receptor numbers. Using a combination of stochastic analysis and Monte Carlo simulations, we determine the time-dependent variation in the mean and variance of synaptic receptor numbers for a variety of initial conditions that simulate fluorescence recovery after photobleaching experiments, and indicate how such data might be used to infer certain properties of the PSD.

Cocaine self-administration produces a persistent increase in dopamine D2 High receptors

Cocaine addicts are reported to have decreased numbers of striatal dopamine D2 receptors. However, in rodents, repeated cocaine administration consistently produces hypersensitivity to the psychomotor activating effects of both indirect dopamine agonists, such as cocaine itself, and importantly, to direct-acting D2 receptor agonists. The current study reports a possible resolution to this long-standing paradox. The dopamine D2 receptor exists in both a low and a high-affinity state, and dopamine exerts its effects via the more functionally relevant high-affinity D2 receptor (D2 High). We report here that cocaine self-administration experience produces a large (approximately 150%) increase in the proportion of D2 High receptors in the striatum with no change in the total number of D2 receptors, and this effect is evident both 3 and 30 days after the discontinuation of cocaine self-administration. Changes in D2 High receptors would not be evident with the probes used in human (and non-human primate) imaging studies. We suggest, therefore, that cocaine addicts and animals previously treated with cocaine may be hyper-responsive to dopaminergic drugs in part because an increase in D2 High receptors results in dopamine supersensitivity. This may also help explain why stimuli that increase dopamine neurotransmission, including drugs themselves, are so effective in producing relapse in individuals with a history of exposure to cocaine.

Are adenosine or adrenergic receptors altered in rat brain following chronic aspartame consumption?

The specific aim of this study was to determine if long‐term aspartame administration in rats would cause a change in brain adrenergic or adenosine receptor densities, as a possible biochemical explanation for our previous results showing memory impairment in these animals. Male Sprague Dawley rats (225 g) received either plain tap water or aspartame in the drinking water (250 mg/kg/day) for 4 months. They were then were sacrificed, brains quickly removed and homogenate preparations made and frozen. Receptor numbers were determined using radioligands to label the receptors. Whole brain preparations, from aspartame‐treated rats showed a statistically significant increase in total apparent adenosine A1 receptor numbers when compared to controls (P&lt;0.05). No significant differences were seen between control and treated animals for any of the adrenergic receptors measured. A trend was seen, however, for the alpha1 and alpha 2 receptors to be slightly decreased in the treated animals. More animals and various brain areas need to be studied to further define any differences. Studies such as these are needed to identify possible CNS effects of aspartame and correlate these with observed changes in memory or learning.

RESEARCH ARTICLE: Up‐regulation of Adenosine Receptors in the Frontal Cortex in Alzheimer's Disease

AbstractAdenosine receptors are G‐protein coupled receptors which modulate neurotransmitter release, mainly glutamate. Adenosine A1 and A2A receptors were studied in post‐mortem human cortex in Alzheimer's disease (AD) and age‐matched controls. Total adenosine A1 receptor number, determined by radioligand binding assay, using [3H]DPCPX, was significantly increased in AD cases in early and advanced stages without differences with the progression of the disease. A significant increase of A1R (37 kDa) levels was also observed by Western blot in early and advanced stages of AD. In addition, increased numbers of adenosine A2A receptors were observed in AD samples as determined by a binding assay using [3H]ZM 241385 as a radioligand and by Western blot. Increased binding and protein expression levels of adenosine receptors were not associated with increased mRNA levels coding A1 and A2A receptors. Finally, increased A1 and A2A receptor‐mediated response was observed. These results show up‐regulation of adenosine A1 and A2A receptors in frontal cortex in AD, associated with sensitization of the corresponding transduction pathways.

Lamina-Specific Abnormalities of NMDA Receptor-Associated Postsynaptic Protein Transcripts in the Prefrontal Cortex in Schizophrenia and Bipolar Disorder

The hypothesis of N-methyl-D-aspartate (NMDA) receptor hypofunction in schizophrenia was initially based on observations that blockade of the NMDA subtype of glutamate receptor by noncompetitive antagonists, such as phencyclidine and ketamine, can lead to clinical symptoms similar to those present in schizophrenia. Recently, glutamate has also been implicated in the pathophysiology of the mood disorders. As impaired NMDA receptor activity may be the result of a primary defect in the NMDA receptors themselves, or secondary to dysfunction in the protein complexes that mediate their signaling, we measured expression of both NMDA subunits and associated postsynaptic density (PSD) proteins (PSD95, neurofilament-light (NF-L), and SAP102) transcripts in the dorsolateral prefrontal cortex in subjects with schizophrenia, bipolar disorder, major depression, and a comparison group using tissue from the Stanley Foundation Neuropathology Consortium. We found decreased NR1 expression in all three illnesses, decreased NR2A in schizophrenia and major depression, and decreased NR2C in schizophrenia. We found no changes of NR2B or NR2D. Receptor autoradiography revealed no alterations in receptor binding in any of the illnesses, indicating no change in total receptor number, but taken with the subunit data suggests abnormal receptor stoichiometry. In the same subjects, PSD95 was unchanged in all three illnesses, while reduced NF-L expression was found in schizophrenia, especially in large cells of layer V. SAP102 expression was reduced in bipolar disorder restricted to small cells of layer II and large cells of layer III in bipolar disorder. These alterations likely reflect altered signaling cascades associated with glutamate-mediated neurotransmission within specific cortical circuits in these psychiatric illnesses.

Dynamic Regulation of Phosphoinositide 3-Kinase-γ Activity and β-Adrenergic Receptor Trafficking in End-Stage Human Heart Failure

Downregulation of beta-adrenergic receptors (betaARs) under conditions of heart failure requires receptor targeting of phosphoinositide 3-kinase (PI3K)-gamma and redistribution of betaARs into endosomal compartments. Because support with a left ventricular assist device (LVAD) results in significant improvement of cardiac function in humans, we investigated the effects of mechanical unloading on regulation of PI3Kgamma activity and intracellular distribution of betaARs. Additionally, we tested whether displacement of PI3Kgamma from activated betaARs would restore agonist responsiveness in failing human cardiomyocytes. To test the role of PI3K on betaAR endocytosis in failing human hearts, we assayed for PI3K activity in human left ventricular samples before and after mechanical unloading (LVAD). Before LVAD, failing human hearts displayed a marked increase in betaAR kinase 1 (betaARK1)-associated PI3K activity that was attributed exclusively to enhanced activity of the PI3Kgamma isoform. Increased betaARK1-coupled PI3K activity in the failing hearts was associated with downregulation of betaARs from the plasma membrane and enhanced sequestration into early and late endosomes compared with unmatched nonfailing controls. Importantly, LVAD support reversed PI3Kgamma activation, normalized the levels of agonist-responsive betaARs at the plasma membrane, and depleted the betaARs from the endosomal compartments without changing the total number of receptors (sum of plasma membrane and early and late endosome receptors). To test whether the competitive displacement of PI3K from the betaAR complex restored receptor responsiveness, we overexpressed the phosphoinositide kinase domain of PI3K (which disrupts betaARK1/PI3K interaction) in primary cultures of failing human cardiomyocytes. Adenoviral-mediated phosphoinositide kinase overexpression significantly increased basal contractility and rapidly reconstituted responsiveness to beta-agonist. These results suggest a novel paradigm in which human betaARs undergo a process of intracellular sequestration that is dynamically reversed after LVAD support. Importantly, mechanical unloading leads to complete reversal in PI3Kgamma and betaARK1-associated PI3K activation. Furthermore, displacement of active PI3K from betaARK1 restores betaAR responsiveness in failing myocytes.

Decreased muscarinic M1 receptor gene expression in the hypothalamus, brainstem, and pancreatic islets of streptozotocin‐induced diabetic rats

The brain neurotransmitters' receptor activity and hormonal pathways control many physiological functions in the body. Acetylcholine (ACh), a major neurotransmitter from autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The objective of the present study was to investigate the changes in the total muscarinic and muscarinic M1 receptor ([(3)H]quinuclidinyl benzilate; QNB) binding and gene expression in the hypothalamus, brainstem, and pancreatic islets of streptozotocin (STZ)-induced diabetic and insulin-treated diabetic rats. In the hypothalamus and brainstem, total muscarinic receptor numbers were increased in diabetic rats with increase in affinity. Hypothalamic and brainstem muscarinic M1 receptors number were decreased in STZ diabetic rats with increase in affinity. In the pancreatic islets, muscarinic M1 receptors of diabetic rats were decreased, with a decrease in affinity. In all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR data also showed a decrease in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. Thus our results suggest that insulin modulates binding parameters and gene expression of total and muscarinic M1 receptors.

The Role of the PDZ Protein GIPC in Regulating NMDA Receptor Trafficking

The NMDA receptor is an important component of excitatory synapses in the CNS. In addition to its synaptic localization, the NMDA receptor is also present at extrasynaptic sites where it may have functions distinct from those at the synapse. Little is known about how the number, composition, and localization of extrasynaptic receptors are regulated. We identified a novel NMDA receptor-interacting protein, GIPC (GAIP-interacting protein, C terminus), that associates with surface as well as internalized NMDA receptors when expressed in heterologous cells. In neurons, GIPC colocalizes with a population of NMDA receptors on the cell surface, and changes in GIPC expression alter the number of surface receptors. GIPC is mainly excluded from the synapse, and changes in GIPC expression do not change the total number of synaptic receptors. Our results suggest that GIPC may be preferentially associated with extrasynaptic NMDA receptors and may play a role in the organization and trafficking of this population of receptors.

Aberrant cerebellar granule cell-specific GABAA receptor expression in the epileptic and ataxic mouse mutant, Tottering

The Tottering (cacna1atg) mouse arose as a consequence of a spontaneous mutation in cacna1a, the gene encoding the pore-forming subunit of the pre-synaptic P/Q-type voltage-gated calcium channel (VGCC, CaV2.1). The mouse phenotype includes ataxia and intermittent myoclonic seizures which have been attributed to impaired excitatory neurotransmission at cerebellar granule cell (CGC) parallel fiber–Purkinje cell (PF–PC) synapses [Zhou YD, Turner TJ, Dunlap K (2003) Enhanced G-protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+-channel mutant mouse, tottering. J Physiol 547:497–507]. We hypothesized that the expression of cerebellar GABAA receptors may be affected by the mutation. Indeed, abnormal GABAA receptor function and expression in the cacna1atg forebrain has been reported previously [Tehrani MH, Barnes EM Jr (1995) Reduced function of gamma-aminobutyric acid A receptors in tottering mouse brain: role of cAMP-dependent protein kinase. Epilepsy Res 22:13–21; Tehrani MH, Baumgartner BJ, Liu SC, Barnes EM Jr (1997) Aberrant expression of GABAA receptor subunits in the tottering mouse: an animal model for absence seizures. Epilepsy Res 28:213–223]. Here we show a deficit of 40.2±3.6% in the total number of cerebellar GABAA receptors expressed (γ2+δ subtypes) in adult cacna1atg relative to controls. [3H]Muscimol autoradiography identified that this was partly due to a significant loss of CGC-specific α6 subunit-containing GABAA receptor subtypes. A large proportion of this loss of α6 receptors was attributable to a significantly reduced expression of the CGC-specific benzodiazepine-insensitive Ro15-4513 (BZ-IS) binding subtype, α6βγ2 subunit-containing receptors. BZ-IS binding was reduced by 36.6±2.6% relative to controls in cerebellar membrane homogenates and by 37.2±3.7% in cerebellar sections. Quantitative immunoblotting revealed that the steady-state expression level of α6 and γ2 subunits was selectively reduced relative to controls by 30.2±8.2% and 38.8±13.1%, respectively, α1, β3 and δ were unaffected. Immunohistochemically probed control and cacna1atg cerebellar sections verified that α6 and γ2 subunit expression was reduced and that this deficit was restricted to the CGC layer. Thus, we have shown that abnormal cerebellar P/Q-type VGCC activity results in a deficit of CGC-specific subtype(s) of GABAA receptors which may contribute to, or may be a consequence of the impaired cerebellar network signaling that occurs in cacna1atg mice.

Two Synergistic Activation Mechanisms of α2β1 Integrin-mediated Collagen Binding

Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, alpha2beta1 integrin. Concomitantly, TPA increases the avidity of alpha2beta1 for collagen and the number of conformationally activated alpha2beta1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the "open" (active) conformation of the inserted domain in the alpha2 subunit (alpha2I). Amino acid residue Glu-336 in alpha2 subunit is proposed to mediate the interaction between alpha2I domain and beta1 subunit. Glu-336 seems to regulate a switch between open and "closed" conformations, since the mutation alpha2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or alpha2beta1 aggregation. Thus, alpha2beta1 integrin avidity is regulated by two synergistic mechanisms, first an alpha2E336-dependent switch to the open alpha2I conformation, and second an alpha2E336-independent mechanism temporally associated with receptor aggregation.

Inhibition of the Dopamine D1 Receptor Signaling by PSD-95

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH(2) terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.

Metabotropic glutamate receptor/phospholipase C system in female rat heart

Glutamate is the main excitatory neurotransmitter in the central nervous system. This amino acid mediates learning and memory processes acting through ionotropic and metabotropic receptor binding. Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that stimulate phospholipase C (PLC) or inhibit adenylyl cyclase (AC). MGluRs have been widely described in CNS. However, little is known about these receptors in peripheral system. The present work describes the mGluR/PLC pathway in membranes from pregnant and non-pregnant rat heart by radioligand binding, Western-blot assays and PLC activity determination. Furthermore, mRNA coding mGluR 1, mGluR 5, αG q/11 and PLCβ 1 was identified by RT-PCR. Binding assays indicated total mGlu receptor numbers of 4.7 ± 0.2 pmol/mg protein and 4.2 ± 1.0 pmol/mg protein in non-pregnant and pregnant rats respectively, and their corresponding K D values were 545.3 ± 85.6 nM and 1062.8 ± 393.6 nM. Western blots revealed bands corresponding to mGluR 1 and mGluR 5 receptors, confirming that these receptors are expressed in heart. The β 1 isoform of PLC, which mediates group I mGluRs (mGluR I) response, was also expressed in rat heart. Moreover, PLC activity was modulated by calcium in a dose-dependent manner. Finally, specific agonists for mGluRs increased the PLC activity and the increase was prevented by specific mGluR antagonists. These results demonstrate the presence of group I mGlu receptors and their functional coupling to the PLC stimulation in female rat heart, suggesting a possible role of mGluR/PLC pathway in this tissue.