Natriuretic Peptides Inhibit Nicotine-Induced Whole-Cell Currents and Catecholamine Secretion in Bovine Chromaffin Cells: Evidence for the Involvement of the Atrial Natriuretic Factor R2 Receptors

Abstract: There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R_1C but not the ANF-R_1A receptor subtype. Evidence for the existence of ANF-R₂ receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R₂-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R₂ receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R₂ receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that this effect of CNP is predominantly mediated by the ANF-R₂ and not the ANF-R_1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCI-induced secretions were affected by natriuretic peptides. In the present study, we report (1) the presence of ANF-R_1C and ANF-R₂ receptor subtypes in bovine chromaffin cells, (2) the inhibition by natriuretic peptides of nicotinic whole-cell currents as well as nicotine-induced catecholamine secretion, (3) the possible mediation of these effects by the ANF-R₂ class of receptors, and (4) the specificity of this inhibition to nicotinic agonists. Because bovine chromaffin cells release ANF, BNP, and CNP together with catecholamines, all three peptides might exert negative feedback regulation of catecholamine secretion in an autocrine manner by interacting with the nondiscriminating ANF-R₂ receptor subtype.     

Introduction to Thematic Minireview Series on Celebrating the Discovery of the Cysteine Loop Ligand-gated Ion Channel Superfamily

The year 2012 marks the 25th anniversary of the discovery of the Cys loop ligand-gated ion channel superfamily of neurotransmitter receptors. This minireview series celebrates this with a series of articles reviewing current information for each of the family members, nicotinic acetylcholine receptors, glycine receptors, GABA_A receptors, serotonin-3 (5-HT₃) receptors, and glutamate-gated chloride ion channels of proteasome invertebrate phyla.     

Mutagenic Analysis of the Intracellular Portals of the Human 5-HT3A Receptor

Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg⁴³⁶ and Arg⁴⁴⁰) within the MA helix of 5-hydroxytryptamine type 3A (5-HT₃A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT₃A receptor model. We probed the majority of residues within the MA helix of the human 5-HT₃A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT₃A receptor portals, which agrees well with the homology model.     

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals

The 5-HT₃A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT₃A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT₃A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.     

Molecular identification of high and low affinity receptors for nicotinic acid

Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G(i)-G protein-coupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G(i)-G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.     

Binding Interactions with the Complementary Subunit of Nicotinic Receptors

The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4β2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)₂(β4)₃. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4β4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.     

In glycine and GABA(A) channels, different subunits contribute asymmetrically to channel conductance via residues in the extracellular domain

Single-channel conductance in Cys-loop channels is controlled by the nature of the amino acids in the narrowest parts of the ion conduction pathway, namely the second transmembrane domain (M2) and the intracellular helix. In cationic channels, such as Torpedo ACh nicotinic receptors, conductance is increased by negatively charged residues exposed to the extracellular vestibule. We now show that positively charged residues at the same loop 5 position boost also the conductance of anionic Cys-loop channels, such as glycine (α1 and α1β) and GABA(A) (α1β2γ2) receptors. Charge reversal mutations here produce a greater decrease on outward conductance, but their effect strongly depends on which subunit carries the mutation. In the glycine α1β receptor, replacing Lys with Glu in α1 reduces single-channel conductance by 41%, but has no effect in the β subunit. By expressing concatameric receptors with constrained stoichiometry, we show that this asymmetry is not explained by the subunit copy number. A similar pattern is observed in the α1β2γ2 GABA(A) receptor, where only mutations in α1 or β2 decreased conductance (to different extents). In both glycine and GABA receptors, the effect of mutations in different subunits does not sum linearly: mutations that had no detectable effect in isolation did enhance the effect of mutations carried by other subunits. As in the nicotinic receptor, charged residues in the extracellular vestibule of anionic Cys-loop channels influence elementary conductance. The size of this effect strongly depends on the direction of the ion flow and, unexpectedly, on the nature of the subunit that carries the residue.     

Sustained Glutamate Receptor Activation Down-regulates GABAB Receptors by Shifting the Balance from Recycling to Lysosomal Degradation

Metabotropic GABA_B receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here, we tested the hypothesis that glutamatergic neurotransmission may regulate GABA_B receptors. We found that application of glutamate to cultured cortical neurons led to rapid down-regulation of GABA_B receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by coactivation of group I metabotropic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca²⁺ channels, and removal of extracellular Ca²⁺ prevented glutamate-induced down-regulation of GABA_B receptors, indicating that Ca²⁺ influx plays a critical role. We further established that glutamate-induced down-regulation depends on the internalization of GABA_B receptors. Glutamate did not affect the rate of GABA_B receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABA_B receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors down-regulates GABA_B receptors by sorting endocytosed GABA_B receptors preferentially to lysosomes for degradation on the expense of recycling. This mechanism may relieve glutamatergic synapses from GABA_B receptor-mediated inhibition resulting in increased synaptic excitability.     

Thymopoietin,a thymic polypeptide,potently interacts at muscle and neuronal nicotinic α-bungarotoxin receptors

Current studies suggest that several distinct populations of nicotinic acetylcholine (ACh) receptors exist. One of these is the muscle-type nicotinic receptors with which neuromuscular nicotinic receptor ligands and the snake toxin alpha-bungarotoxin interact. alpha-Bungarotoxin potently binds to these nicotinic receptors and blocks their function, two characteristics that have made the alpha-toxin a very useful probe for the characterization of these sites. In neuronal tissues, several populations of nicotinic receptors have been identified which, although they share a nicotinic pharmacology, have unique characteristics. The alpha-bungarotoxin-insensitive neuronal nicotinic receptors, which may be involved in mediating neuronal excitability, bind nicotinic agonists with high affinity but do not interact with alpha-bungarotoxin. Subtypes of these alpha-toxin-insensitive receptors appear to exist, as evidenced by findings that some are inhibited by neuronal bungarotoxin whereas others are not. In addition to the alpha-bungarotoxin-insensitive sites, alpha-bungarotoxin-sensitive neuronal nicotinic receptors are also present in neuronal tissues. These latter receptors bind alpha-bungarotoxin with high affinity and nicotinic agonists with an affinity in the microM range. The function of the nicotinic alpha-bungarotoxin receptors are as yet uncertain. Thymopoietin, a polypeptide linked to immune function, appears to interact specifically with nicotinic receptor populations that bind alpha-bungarotoxin. Thus, in muscle tissue where alpha-bungarotoxin both binds to the receptor and blocks activity, thymopoietin also potently binds to the receptor and inhibits nicotinic receptors-mediated function. In neuronal tissues, thymopoietin interacts only with the nicotinic alpha-bungarotoxin site and not the alpha-bungarotoxin-insensitive neuronal nicotinic receptor population. These observations that thymopoietin potently and specifically interacts with nicotinic alpha-bungarotoxin-sensitive receptors in neuronal and muscle tissue, together with findings that thymopoietin is an endogenously occurring agent, could suggest that this immune-related polypeptide represents a ligand for the alpha-bungarotoxin receptors. The function of thymopoietin at the alpha-bungarotoxin receptor is as yet uncertain; however, a potential trophic, as well as other roles are suggested.     

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares approximately 80-90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76-80% identity with the nucleotide and amino acid sequences of these other species. [(3)H]nicotinic acid binding affinity at guinea pig and hamster receptors is similar to that in human (dissociation constant = 121 nM for guinea pig, 72 nM for hamster, and 74 nM for human), as are potencies of nicotinic acid analogs in competition binding studies. Inhibition of forskolin-stimulated cAMP production by nicotinic acid and related analogs is also similar to the activity in the human receptor. Analysis of mRNA tissue distribution for the hamster and guinea pig nicotinic acid receptors shows expression across a number of tissues, with higher expression in adipose, lung, skeletal muscle, spleen, testis, and ovary.     

Two Distinct Allosteric Binding Sites at α4β2 Nicotinic Acetylcholine Receptors Revealed by NS206 and NS9283 Give Unique Insights to Binding Activity-associated Linkage at Cys-loop Receptors

Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (E_max(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding E_max(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.     

The cell adhesion molecule neuroplastin-65 is a novel interaction partner of γ-aminobutyric acid type A receptors

γ-Aminobutyric acid type A (GABA_A) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABA_A receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABA_A receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABA_A receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABA_A receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABA_A receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABA_A receptor subtypes and might contribute to anchoring and/or confining GABA_A receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Endoplasmic Reticulum-associated Degradation Controls Cell Surface Expression of γ-Aminobutyric Acid,Type B Receptors

Metabotropic GABA_B receptors are crucial for controlling the excitability of neurons by mediating slow inhibition in the CNS. The strength of receptor signaling depends on the number of cell surface receptors, which is thought to be regulated by trafficking and degradation mechanisms. Although the mechanisms of GABA_B receptor trafficking are studied to some extent, it is currently unclear whether receptor degradation actively controls the number of GABA_B receptors available for signaling. Here we tested the hypothesis that proteasomal degradation contributes to the regulation of GABA_B receptor expression levels. Blocking proteasomal activity in cultured cortical neurons considerably enhanced total and cell surface expression of GABA_B receptors, indicating the constitutive degradation of the receptors by proteasomes. Proteasomal degradation required Lys⁴⁸-linked polyubiquitination of lysines 767/771 in the C-terminal domain of the GABA_B2 subunit. Inactivation of these ubiquitination sites increased receptor levels and GABA_B receptor signaling in neurons. Proteasomal degradation was mediated by endoplasmic reticulum-associated degradation (ERAD) as shown by the accumulation of receptors in the endoplasmic reticulum upon inhibition of proteasomes, by the increase of receptor levels, as well as receptor signaling upon blocking ERAD function, and by the interaction of GABA_B receptors with the essential ERAD components Hrd1 and p97. In conclusion, the data support a model in which the fraction of GABA_B receptors available for plasma membrane trafficking is regulated by degradation via the ERAD machinery. Thus, modulation of ERAD activity by changes in physiological conditions may represent a mechanism to adjust receptor numbers and thereby signaling strength.     

Up-regulation of GABAB Receptor Signaling by Constitutive Assembly with the K+ Channel Tetramerization Domain-containing Protein 12 (KCTD12)

GABA_B receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA_B receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA_B1a, GABA_B1b, and GABA_B2 form fully functional heteromeric GABA_B(1a,2) and GABA_B(1b,2) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA_B(1a,2) and GABA_B(1b,2) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA_B receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA_B receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA_B receptor-mediated K⁺ current response. In summary, our experiments support that the up-regulation of functional GABA_B receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.     

Opposite Effects of KCTD Subunit Domains on GABAB Receptor-mediated Desensitization

GABA_B receptors assemble from principle and auxiliary subunits. The principle subunits GABA_B1 and GABA_B2 form functional heteromeric GABA_B(1,2) receptors that associate with homotetramers of auxiliary KCTD8, -12, -12b, or -16 (named after their K⁺ channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and -12b generate desensitizing receptor responses while KCTD8 and -16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABA_B2, and a H1 homology domain. KCTD8 and -16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and -12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and -16. In addition, the H2 domains in KCTD8 and -16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and -12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif.     

Dopamine Reduction of GABA Currents in Striatal Medium-sized Spiny Neurons is Mediated Principally by the D<Subscript>1</Subscript> Receptor Subtype

Dopamine modulates voltage- and ligand-gated currents in striatal medium-sized neurons (MSNs) through the activation of D1- and D2-like family receptors. GABA_A receptor-mediated currents are reduced by D1 receptor agonists, but the relative contribution of D₁ or D₅ receptors in this attenuation has been elusive due to the lack of selective pharmacological agents. Here we examined GABA_A receptor-mediated currents and the effects of D1 agonists on MSNs from wildtype and D₁ or D₅ receptor knockout (KO) mice. Immunohistochemical and single-cell RT-PCR studies demonstrated a lack of compensatory effects after genetic deletion of D₁ or D₅ receptors. However, the expression of GABA_A receptor α1 subunits was reduced in D₅ KO mice. At the functional level, whole-cell patch clamp recordings in dissociated MSNs showed that GABA peak current amplitudes were smaller in cells from D₅ KO mice indicating that lack of this receptor subtype directly affected GABA_A-mediated currents. In striatal slices, addition of a D1 agonist reduced GABA currents significantly more in D₅ KO compared to D₁ KO mice. We conclude that D₁ receptors are the main D1-like receptor subtype involved in the modulation of GABA currents and that D₅ receptors contribute to the normal expression of these currents in the striatum. Special issue dedicated to Anthony Campagnoni.     

Promiscuous Dimerization of the Growth Hormone Secretagogue Receptor (GHS-R1a) Attenuates Ghrelin-mediated Signaling

G protein-coupled receptors (GPCRs), such as the ghrelin receptor (GHS-R1a), the melanocortin 3 receptor (MC₃), and the serotonin 2C receptor (5-HT_2C), are well known for their key role in the homeostatic control of food intake and energy balance. Ghrelin is the only known gut peptide exerting an orexigenic effect and has thus received much attention as an anti-obesity drug target. In addition, recent data have revealed a critical role for ghrelin in dopaminergic mesolimbic circuits involved in food reward signaling. This study investigates the downstream signaling consequences and ligand-mediated co-internalization following heterodimerization of the GHS-R1a receptor with the dopamine 1 receptor, as well as that of the GHS-R1a-MC₃ heterodimer. In addition, a novel heterodimer between the GHS-R1a receptor and the 5-HT_2C receptor was identified. Interestingly, dimerization of the GHS-R1a receptor with the unedited 5-HT_2C-INI receptor, but not with the partially edited 5-HT_2C-VSV isoform, significantly reduced GHS-R1a agonist-mediated calcium influx, which was completely restored following pharmacological blockade of the 5-HT_2C receptor. These results combined suggest a potential novel mechanism for fine-tuning GHS-R1a receptor-mediated activity via promiscuous dimerization of the GHS-R1a receptor with other G protein-coupled receptors involved in appetite regulation and food reward. These findings may uncover novel mechanisms of significant relevance for the future pharmacological targeting of the GHS-R1a receptor in the homeostatic regulation of energy balance and in hedonic appetite signaling, both of which play a significant role in the development of obesity.     

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT₁ receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT₁ receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT₁ receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28^(1.32)–Ile-53^(1.57) fragment of TMD1 and Leu-143^(4.40)–Phe-170^(4.67) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C^(1.34)-AT₁ and T33C^(1.37)-AT₁ and TMD4 mutant V169C^(4.66)-AT₁. Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT₁) background, we found that V169C^(4.66)-AT₁ remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C^(4.64)-AT₁ was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30^(1.34) and Thr-33^(1.37), as well as residues Arg-167^(4.64) and Val-169^(4.66) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT₁ receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.