Spinophilin stabilizes cell surface expression of alpha 2B-adrenergic receptors

The third intracellular (3i) loops of the alpha 2A- and alpha 2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the alpha 2A, alpha 2B, and alpha 2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha 2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha 2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha 2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t(1/2) < or = 60 min) apical versus slower (t(1/2) = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha 2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of alpha 2B-AR at the apical surface to approximately 3.6 h and eliminated the rapid phase (0-60 min) of alpha 2B-AR turnover on that surface. Furthermore, we examined alpha 2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp-/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-alpha 2B-AR was accelerated in mouse embryo fibroblasts derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha 2B-AR and presumably all three alpha2-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha 2-ARs to proteins interacting with spinophilin via other domains.     

Protein kinase A phosphorylation of spinophilin modulates its interaction with the alpha 2A-adrenergic receptor (AR) and alters temporal properties of alpha 2AAR internalization

Spinophilin plays critical roles in regulating trafficking and signaling of the alpha(2)-adrenergic receptor (AR) both in vitro and in vivo (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940-1944). In the present study, we demonstrate that protein kinase A (PKA) phosphorylation of spinophilin modulates the spinophilin-alpha(2A)AR interaction to regulate alpha(2A)AR internalization. Activation of PKA by forskolin abolishes the agonist-enhanced interaction between spinophilin and the alpha(2A)AR, and this event can be blocked by Ser --> Ala mutations at the PKA phosphorylation sites of spinophilin. In addition, a Ser --> Asp mutation that mimics the phosphorylated state at the PKA phosphorylation site Ser-177, which is located within the alpha(2A)AR binding region of spinophilin, is sufficient to block the spinophilin-alpha(2A)AR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist-induced internalization of the alpha(2A)AR is accelerated and enhanced, as revealed by both intact cell enzyme-linked immunosorbent assay and quantitative immunofluorescent studies. Furthermore, activation of PKA by forskolin enhances agonist-induced internalization of the alpha(2A)AR in cells expressing wild type spinophilin, but not in cells lacking spinophilin or expressing the spinophilin mutant Sp177D. These results strongly support that PKA phosphorylation of spinophilin is functionally relevant in regulating alpha(2A)AR trafficking. Therefore, modulation of spinophilin-receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates G protein-coupled receptor trafficking.     

The zeta isoform of 14-3-3 proteins interacts with the third intracellular loop of different alpha2-adrenergic receptor subtypes

The alpha2-adrenergic receptors (alpha2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the alpha2AAR (Val217-Ala377), alpha2BAR (Lys210-Trp354), and alpha2CAR (Arg248-Val363) were used as ligands in gel overlay assays. A protein doublet of approximately 30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (alpha2B >/= alpha2C> alpha2A) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the zeta isoform of 14-3-3 proteins. [35S]Met-14-3-3zeta binds to all three native alpha2AR subtypes, assessed using a solid phase binding assay (alpha2A>/=alpha2B> alpha2C), and this binding depends on the presence of the 3i loops. Attenuation of the alpha2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251-266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the alpha2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction.     

Regulated interactions of the alpha 2A adrenergic receptor with spinophilin, 14-3-3zeta,and arrestin 3

The present studies demonstrate that no single stretch of sequence in the third intracellular (3i) loop of the alpha(2A) adrenergic receptor (alpha(2A)-AR) can fully account for its previously described interactions with spinophilin (Richman, J. G., Brady, A. E., Wang, Q., Hensel, J. L., Colbran, R. J., and Limbird, L. E. (2001) J. Biol. Chem. 276, 15003-15008), 14-3-3zeta (Prezeau, L., Richman, J. G., Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 13462-13469), and arrestin 3 (Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836-17842), suggesting that a three-dimensional surface, rather than a linear sequence, provides the basis for these interactions as proposed for 3i loop tethering of the alpha(2A)-AR to the basolateral surface of Madin-Darby canine kidney cells (Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 16331-16336). Sequences at the extreme N-terminal and C-terminal ends of the 3i loop are critical for interaction with spinophilin but not for interaction with 14-3-3zeta or arrestin 3, for which the C-terminal half of the loop is more important. Competition binding for (35)S-labeled alpha(2A)-AR 3i loop binding to glutathione S-transferase (GST)-spinophilin amino acids 151-444 revealed a relative affinity of spinophilin congruent with arrestin > 14-3-3zeta for the unphosphorylated alpha(2A)-AR 3i loop. Agonist occupancy of the alpha(2A)-AR increases receptor association with spinophilin, and arrestin 3 appears to compete for this enrichment. However, when the G protein-coupled receptor kinase 2 substrate sequence was deleted from the 3i loop, arrestin 3 could not compete for the agonist-enriched binding of spinophilin to the mutant alpha(2A)-AR. These data are consistent with a model where sequential or competitive interactions among spinophilin, arrestin, and/or 14-3-3zeta play a role in alpha(2A)-AR functions.     

Mechanisms regulating the cell surface residence time of the alpha 2A-adrenergic receptor

Despite considerable insights concerning the mechanisms regulating short-term agonist-mediated G protein-coupled receptor (GPCR) internalization, little is known about the mechanisms regulating GPCR surface residence over long periods of time. Herein, we experimentally evaluated mechanisms regulating the surface t(1/2) of various alpha(2A)-adrenergic receptor (alpha(2A)AR) structures. The Delta 3i alpha(2A)AR (lacking the third intracellular loop), D79N alpha(2A)AR (impaired G protein coupling), and CAM alpha(2A)AR (enhanced G protein coupling) all exhibited a cell surface alpha(2A)AR turnover in Chinese hamster ovary cells that was faster than that of the wild type (WT). Cell surface receptor turnover could be slowed with ligand occupancy of D79N alpha(2A)AR (agonist or antagonist) and CAM alpha(2A)AR (antagonist only) but not the Delta 3i- or WT alpha(2A)AR. This selective ligand-induced surface stabilization was paralleled by a dramatic ligand-dependent receptor density upregulation for D79N- and CAM alpha(2A)AR structures. Receptors which exhibited surface turnover and density that could be modulated by ligand (D79N and CAM) also demonstrated structural instability, measured by a loss of radioligand binding capacity in detergent solution over time without parallel changes in receptor protein content. In contrast, the shorter surface t(1/2) of the Delta 3i alpha(2A)AR, whose cell surface t(1/2) and steady state density were not altered by ligand occupancy, occurred in the context of a structurally stable receptor in detergent solution. These results demonstrate that changes in receptor structure which alter receptor-G protein coupling (either an increase or decrease) are paralleled by structural instability and ligand-induced surface stabilization. These studies also provide criteria for assessing the structural instability of the alpha(2A)AR that can likely be generalized to all GPCRs.     

Role for the third intracellular loop in cell surface stabilization of the alpha2A-adrenergic receptor.

Previous studies have shown that alpha2A-adrenergic receptor (alpha2A-AR) retention at the basolateral surface of polarized MDCKII cells involves its third intracellular (3i loop). The present studies examining mutant alpha2A-ARs possessing short deletions of the 3i loop indicate that no single region can completely account for the accelerated surface turnover of the Delta3ialpha2A-AR, suggesting that the entire 3i loop is involved in basolateral retention. Both wild-type and Delta3i loop alpha2A-ARs are extracted from polarized Madin-Darby canine kidney (MDCK) cells with 0.2% Triton X-100 and with a similar concentration/response profile, suggesting that Triton X-100-resistant interactions of the alpha2A-AR with cytoskeletal proteins are not involved in receptor retention on the basolateral surface. The indistinguishable basolateral t(1)/(2) for either the wild-type or nonsense 3i loop alpha2A-AR suggests that the stabilizing properties of the alpha2A-AR 3i loop are not uniquely dependent on a specific sequence of amino acids. The accelerated turnover of Delta3i alpha2A-AR cannot be attributed to alteration in agonist-elicited alpha2A-AR redistribution, because alpha2A-ARs are not down-regulated in response to agonist. Taken together, the present studies show that stabilization of the alpha2A-AR on the basolateral surface of MDCKII cells involves multiple mechanisms, with the third intracellular loop playing a central role in regulating these processes.     

Model structures of alpha-2 adrenoceptors in complex with automatically docked antagonist ligands raise the possibility of interactions dissimilar from agonist ligands

Antagonist binding to alpha-2 adrenoceptors (alpha2-ARs) is not well understood. Structural models were constructed for the three human alpha2-AR subtypes based on the bovine rhodopsin X-ray structure. Twelve antagonist ligands (including covalently binding phenoxybenzamine) were automatically docked to the models. A hallmark of agonist binding is the electrostatic interaction between a positive charge on the agonist and the negatively charged side chain of D3.32. For antagonist binding, ion-pair formation would require deviations of the models from the rhodopsin structural template, e.g., a rotation of TM3 to relocate D3.32 more centrally within the binding cavity, and/or creation of new space near TM2/TM7 such that antagonists would be shifted away from TM5. Thus, except for the quinazolines, antagonist ligands automatically docked to the model structures did not form ion-pairs with D3.32. This binding mode represents a valid alternative, whereby the positive charge on the antagonists is stabilized by cation-pi interactions with aromatic residues (e.g., F6.51) and antagonists interact with D3.32 via carboxylate-aromatic interactions. This binding mode is in good agreement with maps derived from a molecular interaction library that predicts favorable atomic contacts; similar interaction environments are seen for unrelated proteins in complex with ligands sharing similarities with the alpha2-AR antagonists.     

Cardiovascular alpha1-adrenoceptor subtypes: functions and signaling

alpha-Adrenoceptors (alpha1AR) are G protein-coupled receptors and include alpha1A, alpha1B, and alpha1D subtypes corresponding to cloned alpha1a, alpha1b, and alpha1d, respectively. alpha1AR mediate several cardiovascular actions of sympathomimetic amines such as vasoconstriction and cardiac inotropy, hypertrophy, metabolism, and remodeling. alpha1AR subtypes are products of separate genes and differ in structure, G protein-coupling, tissue distribution, signaling, regulation, and functions. Both alpha(1A)AR and alpha(1B)AR mediate positive inotropic responses. On the other hand, cardiac hypertrophy is primarily mediated by alpha(1A)AR. The only demonstrated major function of alpha(1D)AR is vasoconstriction. alpha1AR are coupled to phospholipase C, phospholipase D, and phospholipase A2; they increase intracellular Ca2+ and myofibrillar sensitivity to Ca2+ and cause translocation of specific phosphokinase C isoforms to the particulate fraction. Cardiac hypertrophic responses to alpha1AR agonists might involve activation of phosphokinase C and mitogen-activated protein kinase via Gq x alpha1AR subtypes might interact with each other and with other receptors and signaling mechanisms.     

Subtype-selective desensitization of alpha 2-adrenergic receptors. Different mechanisms control short and long term agonist-promoted desensitization of alpha 2C10, alpha 2C4, and alpha 2C2.

We have recently shown that the alpha 2C10 adrenergic receptor (AR) undergoes short term agonist-promoted desensitization, mediated by phosphorylation of sites in the third intracellular loop. There is significant divergence in the third loop amino acid sequences between alpha 2C10 and the other subtypes, alpha 2C4 and alpha 2C2. We therefore explored the mechanisms of alpha 2AR subtype desensitization by expressing each human subtype in Chinese hamster ovary cells and subjecting them to short and long term epinephrine exposures. After 30 min of agonist exposure, alpha 2C10 and alpha 2C2 displayed desensitization characterized by rightward shifts in the curves for epinephrine-mediated inhibition of adenylyl cyclase (EC50 = alpha 2C10, 0.24 +/- 0.02 microM increasing to 1.1 +/- 0.1 microM; alpha 2C2, 1.3 +/- 0.3 increasing to 2.6 +/- 0.3 microM). Coincident with alpha 2C10 and alpha 2C2 desensitizations were decreases in agonist high affinity binding. In contrast, alpha 2C4 underwent no functional desensitization after short term agonist exposure, nor were there any changes in agonist high affinity binding. Agonist-promoted receptor sequestration was clearly greater with alpha 2C10 (approximately 26%) and alpha 2C2 (approximately 35%) as compared to alpha 2C4 (approximately 12%), but such sequestration did not play a significant role in short term desensitization, as blockade with concanavalin A had no effect on desensitization patterns. In contrast to these findings, all alpha 2AR subtypes underwent desensitization after prolonged (24 h) agonist exposure. However, alpha 2C10 and alpha 2C2 displayed substantially more desensitization (approximately 20-fold increase in EC50) as compared to alpha 2C4 (approximately 5-fold increase). The primary mechanism of desensitization during long term agonist exposure was found to be a decrease in the amount of cellular Gi, which was equivalent in magnitude in cells expressing all three subtypes. However, in addition to a decrease in Gi, alpha 2C10 and alpha 2C2 underwent down-regulation of receptor levels during long term agonist exposure, while alpha 2C4 did not. Given that all three subtypes bind endogenous catecholamines with high affinity and inhibit adenylyl cyclase efficiently, the significance of multiple subtypes has heretofore been obscure. Our results show that alpha 2AR undergo subtype-selective desensitization to agonists and suggest that alpha 2AR subtypes may have evolved to meet differing needs for adaptive regulation.     

Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response

The α₂ adrenergic receptor (AR) subtypes are important for blood pressure control. When activated, the α_2A subtype elicits a hypotensive response whereas the α_2B subtype mediates a hypertensive effect that counteracts the hypotensive response by the α_2A subtype. We have previously shown that spinophilin attenuates the α_2AAR-dependent hypotensive response; in spinophilin null mice, this response is highly potentiated. In this study, we demonstrate that spinophilin impedes arrestin-dependent phosphorylation and desensitization of the α_2BAR subtype by competing against arrestin binding to this receptor subtype. The Del301-303 α_2BAR, a human variation that shows impaired phosphorylation and desensitization and is linked to hypertension in certain populations, exhibits preferential interaction with spinophilin over arrestin. Furthermore, Del301-303 α_2BAR-induced ERK signaling is quickly desensitized in cells without spinophilin expression, showing a profile similar to that induced by the wild type receptor in these cells. Together, these data suggest a critical role of spinophilin in sustaining α_2BAR signaling. Consistent with this notion, our in vivo study reveals that the α_2BAR-elicited hypertensive response is diminished in spinophilin deficient mice. In arrestin 3 deficient mice, where the receptor has a stronger binding to spinophilin, the same hypertensive response is enhanced. These data suggest that interaction with spinophilin is indispensable for the α_2BAR to elicit the hypertensive response. This is opposite of the negative role of spinophilin in regulating α_2AAR-mediated hypotensive response, suggesting that spinophilin regulation of these closely related receptor subtypes can result in distinct functional outcomes in vivo. Thus, spinophilin may represent a useful therapeutic target for treatment of hypertension.     

CB1 receptor-G protein association. Subtype selectivity is determined by distinct intracellular domains.

The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C-terminal domain (CB(1)401-417). Guanosine 5'-O-(3-thio)triphosphate binding to G proteins can be stimulated by both peptide CB(1)401-417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol. 53, 504-510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry 38, 3447-3455]. In Chaps-solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB(1) receptor-G alpha(i) association and attenuated the CB(1)401-417-induced inhibition of adenylate cyclase. CB(1)401-417 significantly reduced the CB(1) receptor association with G alpha(i3), but not with G alpha(i1) or G alpha(i2). In contrast, third intracellular loop peptides significantly reduced the CB(1) receptor association with G alpha(i1) and G alpha(i2), but not G alpha(i3). These interactions are specific for the CB(1) receptor because a peptide corresponding to the juxtamembrane C-terminal domain of the CB(2) receptor failed to compete for the association of the CB1 receptor with any of the Gi alpha subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB(1) receptor direct the interaction with specific G protein subtypes.     

Coupling of a mutated form of the human beta 2-adrenergic receptor to Gi and Gs. Requirement for multiple cytoplasmic domains in the coupling process.

We constructed five genes encoding mutant human beta 2-adrenergic receptor sequence (beta 2AR) which contained 12-22 amino acid substitutions with corresponding sequence from the human alpha 2AAR in order to assess the receptor domains involved in Gs versus Gi recognition and coupling. Mutant beta 2AR with substitutions in the N (S1)- and C-terminal (S2) portions of the third intracellular loop, the proximal cytoplasmic tail (S3), and two combinations thereof (S2,3 and S1,2,3), were stably expressed in Chinese hamster fibrobasts (CHW-1102), as were the human beta 2AR and alpha 2AAR at comparable receptor levels. All mutant receptors with S2 substitutions (i.e. S2, S2,3, S1,2,3) were significantly (approximately 85%) uncoupled from Gs. Upon exposure to pertussis toxin, which uncouples receptors from Gi, S1,2,3 exhibited a 526 +/- 99% increase in agonist-stimulated adenylylcyclase activity compared with a 59 +/- 13% increase with the wild type receptor. This enhanced ability of S1,2,3 to interact with Gs following pertussis toxin treatment indicates that, in the absence of toxin exposure, substantial coupling occurs between the mutant receptor and Gi. Mutant beta 2AR bearing only one or two alpha 2AAR-substituted sequences showed no such enhancement. Forskolin-stimulated enzyme activities were increased by pertussis toxin treatment to similar degrees in all clones examined, indicating that the observed effects are confined to the receptor-mediated pathway. In the absence of GTP, competition binding experiments with S1,2,3, beta 2AR and alpha 2AAR revealed that approximately 40-50% of the receptors formed a high affinity binding state for agonist. Pertussis toxin treatment markedly reduced this to approximately 19% with S1,2,3, while having no effect on beta 2AR and completely eliminating high affinity agonist binding to alpha 2AAR. These results suggest that S1,2,3 interacts with Gi as well as Gs, and that receptor:G protein coupling requires the concerted participation of multiple cytoplasmic receptor domains.     

Theoretical study of the electrostatically driven step of receptor-G protein recognition

This study proposes a theoretical model describing the electrostatically driven step of the alpha 1 b-adrenergic receptor (AR)-G protein recognition. The comparative analysis of the structural-dynamics features of functionally different receptor forms, i.e., the wild type (ground state) and its constitutively active mutants D142A and A293E, was instrumental to gain insight on the receptor-G protein electrostatic and steric complementarity. Rigid body docking simulations between the different forms of the alpha 1 b-AR and the heterotrimeric G alpha q, G alpha s, G alpha i1, and G alpha t suggest that the cytosolic crevice shared by the active receptor and including the second and the third intracellular loops as well as the cytosolic extension of helices 5 and 6, represents the receptor surface with docking complementarity with the G protein. On the other hand, the G protein solvent-exposed portions that recognize the intracellular loops of the activated receptors are the N-terminal portion of alpha 3, alpha G, the alpha G/alpha 4 loop, alpha 4, the alpha 4/beta 6 loop, alpha 5, and the C-terminus. Docking simulations suggest that the two constitutively active mutants D142A and A293E recognize different G proteins with similar selectivity orders, i.e., G alpha q approximately equal to G alpha s > G alpha i > G alpha t. The theoretical models herein proposed might provide useful suggestions for new experiments aiming at exploring the receptor-G protein interface.     

Association of the D2 dopamine receptor third cytoplasmic loop with spinophilin, a protein phosphatase-1-interacting protein.

Signaling through D2 class dopamine receptors is crucial to correct brain development and function, and dysfunction of this system is implicated in major neurological disorders such as Parkinson's disease and schizophrenia. To investigate potential novel mechanisms of D2 receptor regulation, the third cytoplasmic loop of the D2 dopamine receptor was used to screen a rat hippocampal yeast two-hybrid library. Spinophilin, a recently characterized F-actin and protein phosphatase-1-binding protein with a single PDZ domain was identified as a protein that specifically associates with this region of D2 receptors. A direct interaction between spinophilin and the D2 receptor was confirmed in vitro using recombinant fusion proteins. The portion of spinophilin responsible for interacting with the D2 third cytoplasmic loop was narrowed to a region that does not include the actin-binding domain, the PDZ domain, or the coiled-coil. This region is distinct from the site of interaction with protein phosphatase-1, and both D2 receptors and protein phosphatase-1 may bind spinophilin at the same time. The interaction is not mediated via the unique 29-amino acid insert in D2long; both D2long and D2short third cytoplasmic loops interact with spinophilin in vitro and in yeast two-hybrid assays. Expression of D2 receptors containing an extracellular hemagglutinin epitope in Madin-Darby canine kidney cells results in co-localization of receptor and endogenous spinophilin as determined by immunocytochemistry using antibodies directed against spinophilin and the HA tag. We hypothesize that spinophilin is important for establishing a signaling complex for dopaminergic neurotransmission through D2 receptors by linking receptors to downstream signaling molecules and the actin cytoskeleton.     

The CXCR1 and CXCR2 receptors form constitutive homo- and heterodimers selectively and with equal apparent affinities

Both homo- and heterodimeric interactions between the CXCR1 and CXCR2 chemokine receptors were observed following co-expression of forms of these receptors in HEK293 cells using assays, including co-immunoprecipitation, single cell imaging of fluorescence resonance energy transfer, cell surface time-resolved fluorescence resonance energy transfer, and bioluminescence resonance energy transfer. These interactions were constitutive and unaffected by the presence of the agonist interleukin 8 and selective as no significant interactions were noted between either the CXCR1 or CXCR2 receptor and the alpha(1A)-adrenoreceptor. Saturation bioluminescence resonance energy transfer indicated that heteromeric interactions between CXCR1 and CXCR2 were of similar affinity as the corresponding homomeric interactions. A novel endoplasmic reticulum trapping strategy demonstrated that these interactions were initiated during protein synthesis and maturation and prior to cell surface delivery. These studies indicated that CXCR1-CXCR2 heterodimers are as likely to form in cells co-expressing these two chemokine receptors as the corresponding homodimers and stand in contrast to previous studies indicating an inability of the CXCR1 receptor to homodimerize or to interact with the CXCR2 receptor (Trettel, F., Di Bartolomeo, S., Lauro, C., Catalano, M., Ciotti, M. T., and Limatola, C. (2003) J. Biol. Chem. 278, 40980-40988).     

NMR structure of the second intracellular loop of the alpha 2A adrenergic receptor: evidence for a novel cytoplasmic helix

A major, unresolved question in signal transduction by G protein coupled receptors (GPCRs) is to understand how, at atomic resolution, a GPCR activates a G protein. A step toward answering this question was made with the determination of the high-resolution structure of rhodopsin; we now know the intramolecular interactions that characterize the resting conformation of a GPCR. To what degree does this structure represent a structural paradigm for other GPCRs, especially at the cytoplasmic surface where GPCR-G protein interaction occurs and where the sequence homology is low among GPCRs? To address this question, we performed NMR studies on approximately 35-residue-long peptides including the critical second intracellular loop (i2) of the alpha 2A adrenergic receptor (AR) and of rhodopsin. To stabilize the secondary structure of the peptide termini, 4-12 residues from the adjacent transmembrane helices were included and structures determined in dodecylphosphocholine micelles. We also characterized the effects on an alpha 2A AR peptide of a D130I mutation in the conserved DRY motif. Our results show that in contrast to the L-shaped loop in the i2 of rhodopsin, the i2 of the alpha 2A AR is predominantly helical, supporting the hypothesis that there is structural diversity within GPCR intracellular loops. The D130I mutation subtly modulates the helical structure. The spacing of nonpolar residues in i2 with helical periodicity is a predictor of helical versus loop structure. These data should lead to more accurate models of the intracellular surface of GPCRs and of receptor-mediated G protein activation.     

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Alpha2A- and alpha2C-adrenergic receptors form homo- and heterodimers: the heterodimeric state impairs agonist-promoted GRK phosphorylation and beta-arrestin recruitment

Dimerization of seven transmembrane-spanning receptors diversifies their pharmacologic and physiologic properties. The alpha(2)-adrenergic receptor (alpha(2)AR) subtypes A and C are both expressed on presynaptic nerves and act to inhibit norepinephrine release via negative feedback. However, in vivo and in vitro studies examining the roles of the two individual alpha(2A)- and alpha(2C)AR subtypes are not readily reconciled. We tested the hypothesis that the receptors form homo- and heterodimers and that the alpha(2A)-alpha(2C) heterodimer has unique properties. SDS-PAGE of epitope-tagged receptors revealed potential oligomers including dimers. BRET of live HEK-293 cells transfected with the subtypes fused to Rluc or YFP revealed that both subtypes form dimers and the heterodimer. A lower BRET(50) for the alpha(2A)-alpha(2C) heterodimer (0.79 +/- 0.20) compared to that of the alpha(2A) or alpha(2C) homodimer (2.331 +/- 0.44 or 3.67 +/- 0.69, respectively) suggests that when both subtypes are expressed, there is a greater likelihood that the two receptors will form the heterodimer than homodimers. Co-immunoprecipitation studies confirmed homo- and heterodimer formation. The presence of the alpha(2C)AR within the heterodimer resulted in a marked reduction in the level of GRK2-mediated alpha(2A)AR phosphorylation, which was accompanied by a qualitative attenuation of beta-arrestin recruitment. Signaling of the alpha(2A)-alpha(2C) heterodimer to the beta-arrestin-dependent activation of Akt was decreased compared to that of the alpha(2A)AR homodimer, while p44/p42 MAP kinase activation was unaffected. Thus, the alpha(2C)AR alters alpha(2A)AR signaling by forming oligomers, and these complexes, which appear to be preferred over the homodimers, should be considered a functional signaling unit in cells in which both subtypes are expressed.     

激动剂作用下α1-肾上腺素受体亚型在HEK293A细胞中的分布变化

为了明确α1-肾上腺素受体（α1-adrenergic receptor,α1-AR）三种亚型在人胚胎肾（human embryonic kidney,HEK）293A细胞株中的分布特点,及其在激动剂作用下在细胞内的定位改变,本研究采用放射配体结合实验、实时荧光共聚焦成像和Western blot方法检测α1-AR三种亚型在细胞中的定位及蛋白质表达的变化。结果发现：（1）α1-AR三种亚型在HEK293A细胞株转染效率相同,均达90％以上。三株细胞的粗制膜上α1B-AR表达量最高,α1D-AR最低,α1A-AR居中,但三者的解离常数（配）相等;（2）在无激动剂作用时,α1A-AR均匀地分布在HEK293A细胞的胞膜和胞浆,α1B-AR主要位于胞膜,而α1D-AR则主要分布在胞浆中：（3）用α1-AR激动剂苯‘肾上腺素（phenylephrine,PE）刺激细胞1h后,α1A-和α1B-AR在胞膜上分布明显减少,而在胞浆中分布增加,其中α1B-AR变化更为显著,α1D-AR的分布在PE作用下无明显变化。以上结果提示,在激动剂作用下,α1-AR二种亚型在HEK293A细胞中的定位特点和分布变化各有不同。     

Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-halpha7 cells by activating neuronal nicotinic acetylcholine receptors

(-)-Methadone acts as an agonist at opioid receptors. Both (+)- and (-)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing alpha7 and alpha3* nAChR subtypes and SH-EP1-halpha7 cells heterologously expressing human alpha7 nAChRs. Methadone potently inhibited binding of [3H]methyllycaconitine to alpha7 nAChRs and that of [3H]epibatidine to alpha3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH-SY5Y cells. Nicotine and both (+)- and (-)-methadone evoked increases in [Ca2+]i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the alpha7 selective antagonist methyllycaconitine, suggesting effects of methadone as alpha7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl2, ryanodine and xestospongin-c implicates voltage-operated Ca2+ channels and intracellular Ca2+ stores as downstream modulators of elevated [Ca2+]i. Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs.