Chris Hague

Heterodimerization of G Protein-Coupled Receptors: Specificity and Functional Significance

G protein-coupled receptors (GPCRs) are cell surface receptors that mediate physiological responses to a diverse array of stimuli. GPCRs have traditionally been thought to act as monomers, but recent evidence suggests that GPCRs may form dimers (or higher-order oligomers) as part of their normal trafficking and function. In fact, certain GPCRs seem to have a strict requirement for heterodimerization to attain proper surface expression and functional activity. Even those GPCRs that do not absolutely require heterodimerization may still specifically associate with other GPCR subtypes, sometimes resulting in dramatic effects on receptor pharmacology, signaling, and/or internalization. Understanding the specificity and functional significance of GPCR heterodimerization is of tremendous clinical importance since GPCRs are the molecular targets for numerous therapeutic drugs.

Fluorescence changes reveal kinetic steps of muscarinic receptor-mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels

G protein–coupled receptors initiate signaling cascades. M1 muscarinic receptor (M1R) activation couples through Gαq to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2). Depletion of PIP2 closes PIP2-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M1R activation, M1R/Gβ interaction, Gαq/Gβ separation, Gαq/PLC interaction, and PIP2 hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M1R activation (<100 ms) and M1R/Gβ interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Gαq/Gβ separation and Gαq/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP2 hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Gαq/PLC interaction. Evidently, channel release of PIP2 and closure are rapid, and the availability of active PLC limits the rate of M current suppression.

Heterodimers of α1B and α1D-adrenergic receptors form a single functional entity

Heterologous expression of α1D-adrenergic receptors (α1D-ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with α1B-ARs promotes surface localization of α1D-ARs. Here, we report that the α1B-/α1D-AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of α1B-ARs and epitope-tagged α1D-ARs in both human embryonic kidney 293 and DDT1MF-2 cells, α1D-AR binding sites were not detectable with the α1D-AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione (BMY 7378), despite the ability to detect α1D-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the α1B-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in α1B/α1D-AR-expressing cells, revealing that α1D-ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in α1B/α1D-AR-coexpressing cells. Stable coexpression of an uncoupled mutant α1B-AR (Δ12) with α1D-ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that α1B/α1D-AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why α1D-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with α1B-ARs.

Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the α1A-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Gα subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Gα. Combining our data with a predictive Class A GPCR/Gα model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Gα. Such an interaction could promote opening of switch II of Gα to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Blood pressure is regulated by an α1D-adrenergic receptor/dystrophin signalosome

Hypertension is a cardiovascular disease associated with increased plasma catecholamines, overactivation of the sympathetic nervous system, and increased vascular tone and total peripheral resistance. A key regulator of sympathetic nervous system function is the α1D-adrenergic receptor (AR), which belongs to the adrenergic family of G-protein-coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate α1D-ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, α1D-AR KO mice display a hypotensive phenotype and are resistant to salt-induced hypertension. Unfortunately, little information exists about how this important GPCR functions because of an inability to obtain functional expression in vitro. Here, we identified the dystrophin proteins, syntrophin, dystrobrevin, and utrophin as essential GPCR-interacting proteins for α1D-ARs. We found that dystrophins complex with α1D-AR both in vitro and in vivo to ensure proper functional expression. More importantly, we demonstrate that knock-out of multiple syntrophin isoforms results in the complete loss of α1D-AR function in mouse aortic smooth muscle cells and abrogation of α1D-AR-mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with α1D-ARs to create a functional signalosome, which is essential for α1D-AR regulation of vascular tone and blood pressure.

The Role of Norepinephrine in Differential Response to Stress in an Animal Model of Posttraumatic Stress Disorder

BACKGROUND Posttraumatic stress disorder (PTSD) is a prevalent psychiatric disorder precipitated by exposure to extreme traumatic stress. Yet, most individuals exposed to traumatic stress do not develop PTSD and may be considered psychologically resilient. The neural circuits involved in susceptibility or resiliency to PTSD remain unclear, but clinical evidence implicates changes in the noradrenergic system. METHODS An animal model of PTSD called Traumatic Experience with Reminders of Stress (TERS) was developed by exposing C57BL/6 mice to a single shock (2 mA, 10 sec) followed by exposure to six contextual 1-minute reminders of the shock over a 25-day period. Acoustic startle response (ASR) testing before the shock and after the last reminder allowed experimenters to separate the shocked mice into two cohorts: mice that developed a greatly increased ASR (TERS-susceptible mice) and mice that did not (TERS-resilient mice). RESULTS Aggressive and social behavioral correlates of PTSD increased in TERS-susceptible mice but not in TERS-resilient mice or control mice. Characterization of c-Fos expression in stress-related brain regions revealed that TERS-susceptible and TERS-resilient mice displayed divergent brain activation following swim stress compared with control mice. Pharmacological activation of noradrenergic inhibitory autoreceptors or blockade of postsynaptic α(1)-adrenoreceptors normalized ASR, aggression, and social interaction in TERS-susceptible mice. The TERS-resilient, but not TERS-susceptible, mice showed a trend toward decreased behavioral responsiveness to noradrenergic autoreceptor blockade compared with control mice. CONCLUSIONS These data implicate the noradrenergic system as a possible site of pathological and perhaps also adaptive plasticity in response to traumatic stress.

Syntrophins regulate α1D-adrenergic receptors through a PDZ domain-mediated interaction

To find novel cytoplasmic binding partners of the α1D-adrenergic receptor (AR), a yeast two-hybrid screen using the α1D-AR C terminus as bait was performed on a human brain cDNA library. α-Syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an α1D-AR-interacting protein. α-Syntrophin specifically recognized the C terminus of α1D- but not α1A- or α1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms α, β1, and β2 but not γ1 or γ2 showed strong selective interactions with the α1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length α1D- but not α1A- or α1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the α1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the α1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased α1D-AR binding and protein expression. In addition, stable overexpression of α-syntrophin significantly increased α1D-AR protein expression and binding but did not affect those with a mutated PDZ-interacting motif, suggesting that syntrophin plays an important role in maintaining receptor stability by directly interacting with the receptor PDZ-interacting motif. This direct interaction may provide new information about the regulation of α1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.

Subtype-selective noncompetitive or competitive inhibition of human α1-Adrenergic receptors by ρ-TIA

The 19-amino acid conopeptide (ρ-TIA) was shown previously to antagonize noncompetitively α1B-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human α1-AR subtypes (α1A, α1B, and α1D). Radioligand binding assays showed that ρ-TIA was 10-fold selective for human α1B-over α1A- and α1D-ARs. As observed with hamster α1B-ARs, ρ-TIA decreased the number of binding sites (Bmax) for human α1B-ARs without changing affinity (KD), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, ρ-TIA had opposite effects at human α1A-ARs and α1D-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. ρ-TIA reduced maximal NE-stimulated [3H]inositol phosphate formation in HEK293 cells expressing human α1B-ARs but competitively inhibited responses in cells expressing α1A- or α1D-ARs. Truncation mutants showed that the amino-terminal domains of α1B- or α1D-ARs are not involved in interaction with ρ-TIA. Alanine-scanning mutagenesis of ρ-TIA showed F18A had an increased selectivity for α1B-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at α1B-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus ρ-TIA noncompetitively inhibits α1B-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at α1B-ARs.

α-Dystrobrevin-1 recruits α-catulin to the α1D-adrenergic receptor/dystrophin-associated protein complex signalosome

α1D-Adrenergic receptors (ARs) are key regulators of cardiovascular system function that increase blood pressure and promote vascular remodeling. Unfortunately, little information exists about the signaling pathways used by this important G protein-coupled receptor (GPCR). We recently discovered that α1D-ARs form a “signalosome” with multiple members of the dystrophin-associated protein complex (DAPC) to become functionally expressed at the plasma membrane and bind ligands. However, the molecular mechanism by which the DAPC imparts functionality to the α1D-AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α1D-AR signalosome, we performed an extensive proteomic analysis on each member of the DAPC. Bioinformatic analysis of our proteomic data sets detected a common interacting protein of relatively unknown function, α-catulin. Coimmunoprecipitation and blot overlay assays indicate that α-catulin is directly recruited to the α1D-AR signalosome by the C-terminal domain of α-dystrobrevin-1 and not the closely related splice variant α-dystrobrevin-2. Proteomic and biochemical analysis revealed that α-catulin supersensitizes α1D-AR functional responses by recruiting effector molecules to the signalosome. Taken together, our study implicates α-catulin as a unique regulator of GPCR signaling and represents a unique expansion of the intricate and continually evolving array of GPCR signaling networks.

Differential regulation of GPR54 transcription by specificity protein-1 and partial estrogen response element in mouse pituitary cells

Precise spatial and temporal expression of the recently identified G-protein coupled receptor GPR54 is critical for proper reproductive function and metastasis suppression. However, regulatory factors that control GPR54 expression remain unknown. Thus, the identification of these cis-acting DNA elements can provide insight into the role of GPR54 in reproduction and cancer. Using luciferase reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we demonstrate that three SP1 sites and a partial estrogen response element modulate mouse GPR54 (mGPR54) promoter activity. Supporting experiments show transcription factor SP1 binds directly to the mGPR54 promoter region and activates gene expression. In conclusion, these novel findings now identify factors that regulate activity of the mGPR54 promoter, and these factors are highly conserved across multiple mammalian species.