Abstract
Functional and structural data from G protein-coupled receptors (GPCR) predict that transmembrane-domain (TM)2 is adjacent to TM7 within the GPCR structure, and that within this interface a conserved aspartate in TM2 and a conserved asparagine in TM7 exist in close proximity. Mutation at this D79(TM2)-N422(TM7) interface in the α2A-adrenergic receptor (α2AAR) affects not only receptor activation but also cell-surface residence time and conformational stability. Mutation at TM2(D79N) reduces allosteric modulation by Na+ and receptor activation more dramatically than affecting cell-surface receptor turnover and conformational stability, whereas mutation at TM7(N422D) creates profound conformational instability and more rapid degradation of receptor from the surface of cells despite receptor activation and allosteric modulation properties that mirror a wild-type receptor. Double mutation of TM2 and 7(D79N/N422D) reveals phenotypes for receptor activation and conformational stability intermediate between the wild-type and singly mutated α2AAR. Additionally, the structural placement of a negative charge at this TM2/TM7 interface is necessary but not sufficient for receptor structural stability, because mislocalization of the negative charge in either the D79Eα2AAR (which extends the charge out one methylene group) or the D79N/N422Dα2AAR (placing the charge in TM7 instead of TM2) results in conformational lability in detergent solution and more rapid cell-surface receptor clearance. These studies suggest that this interface is important in regulating receptor cell-surface residence time and conformational stability in addition to its previously recognized role in receptor activation.
The α2-adrenergic receptors (α2AR) are members of the superfamily of seven transmembrane spanning G protein-coupled receptors (GPCR) and are coupled via Gi/Go proteins to multiple effectors in native cells such as inhibition of adenylyl cyclase (Limbird, 1988), suppression of voltage-gated Ca2+ currents (Horn and McAffee, 1980), activation of receptor operated K+currents (Morita and North, 1981), and stimulation of the mitogen-activated protein (MAP) kinase cascade (Richman and Regan, 1998).
Cell-surface receptor expression is necessary for extracellular to intracellular communication mediated by circulating hormones. Mechanisms regulating the long-term cell-surface residence time of GPCRs are poorly understood. For the α2AAR, the presence of the third intracellular loop (Edwards and Limbird, 1999) as well as conformational stability (Wilson and Limbird, 2000) contributes to maintaining long-term (approximately 12–14 hours) cell-surface receptor residence time, albeit through differing mechanisms. Although conformational stability likely contributes to maintaining or permitting cell-surface receptor expression, sites critical for maintaining conformational stability within GPCR structure are only beginning to be elucidated. One such site apparently critical for regulating α2AAR conformational stability is the highly conserved D79 residue, located in a topologically homologous position in all amine-binding GPCR in TM2 (Wilson and Limbird, 2000).
The three-dimensional structural characterization of bacteriorhodopsin and subsequently bovine rhodopsin (Unger et al., 1997; Palczewski et al., 2000), in conjunction with intentional mutagenesis strategies coupled with functional studies (Mizobe et al., 1996; Gether and Kobilka, 1998), has led to a proposed arrangement of the seven helices of GPCR within the bilayer. For amine-binding GPCR, a hydrogen bonding network consisting of at least a conserved aspartate in transmembrane-domain 2 (TM2) and an asparagine in TM7 is postulated to serve as a critical link between agonist occupancy and G protein activation. An exception of nature is the gonadotropin releasing hormone (GnRH) receptor, where these two residues are interchanged such that there exists an asparagine in TM2 and an aspartate in TM7 (Zhou et al., 1994). Exchanging these residues between TM2 and TM7 in the GnRH receptor (creating the N87D/D318N GnRH-R) results in a receptor structure that retains high affinity agonist binding and coupling to G proteins, albeit not to the same extent as the wild-type receptor. A single mutation, N87D in TM2 of the GnRH receptor, however, leads to loss of G protein coupling, presumably because of disruption of this hydrogen bonding network, which serves to regulate receptor activation (Zhou et al., 1994). For the GnRH receptor, this interface can also affect receptor expression in transfected cells (Flanagan et al., 1999). An analogous interaction between an aspartate in TM2 and an asparagine in TM7 has been demonstrated for the 5HT2A-receptor (Zhou et al., 1994) and for the μ-opioid receptor (Xu et al., 1999). In contrast, complementary TM2/TM7 interface mutation in the cannabinoid CB1 receptor does not restore receptor-mediated potentiation of inwardly rectifying potassium channel current or receptor internalization (Roche et al., 1999), indicating greater complexity of this predicted charge relay system or lack of generality among all GPCR subclasses. Additionally, the recent crystal structure of rhodopsin reveals that Asp83 (TM2) and Asn302 (TM7) are probably too far apart for a direct hydrogen bonding interaction although they may be bridged by a water molecule, revealing a proximity between TM2 and TM7 in GPCR structure (Palczewski et al., 2000).
With regards to the α2AAR, previous studies have implicated several consequences of mutating this highly conserved aspartate (D79) in TM2 including: 1) changes in allosteric modulation of receptor conformation by monovalent cations, such as Na+ (Horstman et al., 1990; Neve et al., 1991;Kong et al., 1993; Tian and Deth, 1993), 2) altered cell surface residence time of the α2AAR, and 3) diminished conformational/structural stability of the receptor, manifest by loss of functional binding capacity in detergent preparations without parallel changes in receptor protein (Wilson and Limbird, 2000). The present studies explored the role of the conserved TM2(D79)/TM7(N422) interface in the α2AAR and evaluated what role this interface plays in regulating conformational stability, cell-surface residence time and multiple functional properties of the α2AAR.
Materials and Methods
Molecular Modeling.
To generate a schematic of the proposed proximity of D79 in TM2 and N422 in TM7 of the α2AAR, molecular modeling was used to generate a simplified model of this receptor. De novo modeling of the α2AAR was performed via e-mail using SWISS MODEL in the GPCR mode ( http://www.expasy.ch/swissmod/SWISS-MODEL.html ). Briefly, the predicted transmembrane domain spans for the α2AAR were entered into the program using the human β2-AR as the template for modeling. SWISS MODEL then generated models with ProModII and conducted energy minimization with Gromos 96 (Peitsch et al., 1996; Ghex et al., 1999). Swiss-PdbViewer was then used to analyze and visualize the results (Fig. 1).
DNA Reagents.
Porcine hemagglutinin (HA)-tagged wild-type, D79N, D79E, and D79Q α2AAR have been described previously (Ceresa and Limbird, 1994). The N422D mutation was engineered simultaneously into the wild-type and D79N backbones in the pCMV4 mammalian expression vector using QuickChange Site-Directed Mutagenesis (Stratagene, La Jolla, CA). Two complementary oligonucleotides generating the N422D mutation with an additionalBamHI site to facilitate screening (antisense, 5′-GTAGATAACCGGATCCAGCGAGCTGTTGCAGTAG-3′; sense, 5′-CTACTGCAACAGCTCGCTGGATCCGGTTATCTAC-3′) were used in polymerase chain reactions according to the manufacturers instructions, except that 10% dimethyl sulfoxide was used in the reaction to facilitate extension of the template. Colonies were screened via BamHI digest of minipreps and the entire coding region of the N422D and D79N/N422D mutants was then sequenced with33P-thermosequenase-cycle sequencing.
Cell Culture and Transfection.
EcR-CHO (Chinese hamster ovary cells engineered for the ecdysone-inducible expression system obtained from Invitrogen, (Carlsbad, CA) cells were maintained in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and 100 U/ml penicillin G sodium with 100 μg/ml streptomycin sulfate (pen-strep). Human embryonic kidney (HEK) 293 cells were maintained in minimal essential medium supplemented with 10% FCS plus pen-strep. EcR-CHO and HEK293 cells were plated the day before transfection at a density of 2 × 106cells per well of a six-well plate or 35-mm dish. Cells were transiently transfected with the use of FuGENE-6 (Boehringer Mannheim) according to the manufacturer's instructions. Cells were assayed approximately 48 h after transfection.
Guanine Nucleotide Sensitivity of Radiolabeled Agonist Binding as a Measure of α2AAR-G protein Coupling.
To evaluate the ability of Gpp(NH)p, a hydrolysis-resistant GTP analog, to modulate radiolabeled agonist binding, membranes from HEK 293 cells transiently expressing receptor were lysed in hypotonic lysis buffer [15 mM HEPES, 5 mM, EDTA, 5 mM EGTA, pH 8.0 (with the addition of 10 U/ml aprotinin and 0.1 mM PMSF)]. Cells were scraped on ice into ice-cold hypotonic lysis buffer and passaged five times through a 25-gauge needle on ice and centrifuged for 15 min at 40,000g, followed by resuspension in lysis buffer and recentrifugation. Membranes were then resuspended in 50 mM Tris-HCl, 10 mM MgCl2, and 5 mM EGTA, pH 8.0. Incubations (100 μl) containing membranes, 0.9 nMpara-[125I]iodoclonidine ([125I]PIC) agonist radioligand (approximately 160,000 cpm/100-μl incubation) and none (control) or increasing concentrations of Gpp(NH)p were incubated for 30 min at 25°C. Reactions were terminated via vacuum filtration using a Brandel Cell Harvester and ice-cold 25 mM glycylglycine, pH 7.6. Filters were then counted in a Beckman 4000 gamma counter (Beckman Instruments, Palo Alto, CA).
Assessment of Allosteric Modulation of α2AAR Conformation by Na+.
Epinephrine was evaluated as a competitor for [3H]RX821002, a radiolabeled antagonist, in the presence or absence of Na+(Horstman et al., 1990; Ceresa and Limbird, 1994; Lakhlani et al., 1997). Transiently transfected HEK 293 cells expressing receptor were lysed, centrifuged, resuspended, and recentrifuged as outlined above. Membranes were then resuspended in a small volume of lysis buffer using a 25-gauge needle. A small volume of membranes was then added to a binding reaction consisting of 25 mM HEPES, 40 mM glycylglycine, 100 mM NaCl or N-methyl d-glucamine chloride, and 5 mM EDTA, pH 8.0 (final volume, 250 μl) and [3H]RX821002 with none (control) or various concentrations of epinephrine (competitor). Reactions proceeded at 25°C for 30 min and were terminated as above. Filters were counted in Aquasol (Packard, Meriden, CT) in a Packard 1600 TR scintillation counter.
Mitogen-Activated Protein Kinase Assay.
The ability of receptor to activate MAP kinase was evaluated in HEK 293 cells transiently expressing α2AAR, WT, or mutant structures. Cells were serum starved overnight starting 24 after transfection. The following day, response to various concentrations of epinephrine (in serum-free medium) was examined for 2 min using one transfected 35-mm dish per condition. The agonist-containing medium was then aspirated and the incubation was terminated by scraping cells into 100 μl SDS-sample buffer consisting of 62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue (with the addition of 2 mM Na-orthovanadate). This lysate was passaged twice through a 25-gauge needle and then placed on ice. Samples were then sonicated for 30 s and heated at 95°C for 5 min followed by centrifugation at 13,000g for 5 min. An aliquot (40 μl) was then subjected to 10% SDS-PAGE followed by Western analysis for using both anti-active and anti-total MAP kinase antibodies as described previously (Schramm and Limbird, 1999).
Assessment of Ligand-Dependent Receptor Density Up-Regulation.
To assess the effect of agonist or antagonist occupancy on steady-state α2AAR density, EcR-CHO cells transiently expressing WT or mutant α2AAR were incubated in serum-free medium containing 0.1 mM ascorbate alone or ascorbate with 100 μM epinephrine or 10 μM idazoxan for 16 to 24 h. Cells were then washed three times with phosphate-buffered saline, pH 7.4, prewarmed to 37°C to facilitate removal of ligand. Triplicate wells of a six-well plate were then scraped and pooled in 1.2 ml of ice-cold buffer consisting of 25 mM glycylglycine, 40 mM HEPES, 5 mM EDTA, 5 mM EGTA, pH 8.0, 10 U/ml aprotinin, and 0.1 mM PMSF and homogenized with five up/down strokes of a 25-gauge needle on ice. Total cell lysate was then subjected to binding analysis using a saturating concentration of [3H]RX821002 (10 nM). Nonspecific binding was defined as that binding detected in the presence of 10 μM phentolamine. Samples were normalized for milligrams of protein using a protein assay kit (Bio-Rad, Hercules, CA) as outlined above.
Assessment of Cell Surface Receptor Turnover.
To assess cell surface receptor turnover, receptors were first “up-regulated” by overnight treatment with idazoxan to increase the sensitivity of the assay for the mutant receptors (Fig. 3C). After overnight treatment, cells were washed three times with 37°C PBS to wash out the idazoxan, twice on ice with 4°C PBS, and biotinylated at 4°C with 1 mg/ml sulfosuccinimidobiotin (sulfo-NHS-biotin; Pierce Chemical, Rockford, IL) in PBS. Cells were then transferred to 37°C serum free medium with or without receptor ligand (10 μM idazoxan or 100 μM epinephrine in the presence of 100 μM ascorbate) and placed in a 37°C, 5% CO2 incubator. At the time points indicated in Fig. 3A, medium was aspirated from the cells and duplicate wells of a six-well plate were placed on ice and scraped into 500 μl/well of 4 mg/ml dodecyl-β-d-maltoside, 0.8 mg/ml cholesteryl hemisuccinate, 20% glycerol, 25 mM glycylglycine, 20 mM HEPES, 100 mM NaCl, 5 mM EGTA, pH 8.0, 0.1 mM PMSF, and 10 U/ml aprotinin (called DβM/CHS extraction buffer), pooled and passaged five times on ice through a 25-gauge needle. Cellular debris was cleared from solubilized protein via centrifugation at 13,000 rpm in a Microfuge at 4°C for 30 min. This supernatant, referred to as the detergent-solubilized preparation, was then incubated with 50 μl of streptavidin-agarose overnight at 4°C on an inversion wheel. The streptavidin-agarose was washed twice with 0.5 mg/ml dodecyl-β-d-maltoside, 0.1 mg/ml cholesteryl hemisuccinate in 25 mM glyclyglycine, 20 mM HEPES, 100 mM NaCl, 5 mM EDTA, pH 8.0 (called DβM/CHS wash buffer), and then eluted into 150 μl of SDS sample buffer with 15 min at 90°C. These samples were then subjected to SDS-PAGE and transferred to nitrocellulose in a buffer containing 10 mM CAPS, pH 11.0, and 10% methanol for 1.2 h at 1 A. Nitrocellulose was blocked in Tris-buffered saline with 1% Tween 20 containing 5% nonfat dry milk for 1 h at room temperature. HA-tagged receptor was then detected by blotting with a 1:1000 dilution of HA.11 primary antibody (Covance Research Products, Berkeley, CA) in blocking buffer followed by anti-mouse horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ). For semiquantification of Western analyses, films were digitized by scanning into Adobe Photoshop (Adobe Systems, Mountain View CA) and analyzed with NIH image software.
Assessment of Stability of Receptor Binding and Receptor Protein in Detergent-Solubilized Preparations.
Transiently transfected COS M6 cells expressing wild-type or mutant α2AAR were rinsed with PBS 48 h after transfection. Cells were then biotinylated at room temperature with 1 mg/ml sulfosuccinimidobiotin in PBS, as above. The biotinylation solution was then aspirated and cells were scraped on ice into ice-cold 15 mM HEPES, 5 mM EDTA, and 5 mM EGTA, pH 7.6 (with the addition of 10 U/ml aprotinin, 0.1 mM PMSF, 1 mg/ml soybean trypsin inhibitor, and 1 mg/ml leupeptin) and passaged five times up/down through a 25-gauge needle on ice. Lysates were then centrifuged at 40,000g for 15 min at 4°C. Pellets were resuspended on ice into DβM/CHS extraction buffer (with the addition of 10 U/ml aprotinin, 0.1 mM PMSF, 1 mg/ml soybean trypsin inhibitor and 1 mg/ml leupeptin). Receptor was solubilized by 10 up/down strokes in a Teflon/glass homogenizer on ice. Cellular debris was cleared from solubilized protein by centrifugation at 13,000 rpm in a Microfuge for 30 min at 4°C. The supernatant fraction was defined as the detergent-solubilized receptor. To assess receptor stability over time, enough detergent solubilized protein was added to the binding reactions to achieve 0.25–0.5 pmol of bound receptor at time 0 (immediately after solubilization and clearance from cellular debris). Change in functional binding capacity was followed as a function of time by keeping the detergent-solubilized receptors at 25°C and assaying the same volume of solubilized preparation per binding reaction at different time points (Wilson and Limbird, 2000). Specifically, the detergent-solubilized preparation was then warmed to 25°C. At the given time points, after incubation at 25°C, aliquots were removed and incubated with 40 μl streptavidin-agarose and 7.5 nM [3H]yohimbine in DβM/CHS wash buffer (total reaction volume, 500 μl) at 4°C on an inversion wheel for 1 to 1.5 h. Beads were then washed twice with DβM/CHS wash buffer. The remaining beads were then directly added to scintillation cocktail (Aquasol) and counted on a Packard scintillation counter. The stability of receptor protein in these same samples was confirmed by Western analysis of the HA epitope in the α2AAR proteins using the HA.11 antibody, as described above.
Results
To assess retrograde communication (G→R) between G proteins and WT and mutant α2A-AR structures, we examined the ability of Gpp(NH)p to decrease receptor affinity for agonists, measured by the loss of trapability of the [125I]PIC radiolabeled agonist as a function of increasing concentrations of the hydrolysis-resistant GTP analog, Gpp(NH)p (Ceresa and Limbird, 1994). As Gpp(NH)p concentrations are increased, the fraction of receptors in the higher affinity R-G complex (K app ≅ 0.4 nM) is diminished, as is the fraction of receptors that can be detected using the [125I]PIC agonist radioligand (Baron and Siegel, 1990). The lower affinity interactions [R + G-Gpp(NH)p] with the receptor are not trapable using [125I]PIC as the radioligand (Baron and Siegel, 1990; Ceresa and Limbird, 1994;Keefer et al., 1994) (Fig. 1C, schematic). As shown in Fig. 1C, more than 80% of the WT [125I]PIC-specific binding detected is eliminated when incubations contain 100 μM Gpp(NH)p, consistent with the interpretation that the [125I]PIC binding assay predominantly detects radioligand binding to high-affinity R-G complexes. Similar findings and interpretations have been obtained for radiolabeled agonist-binding to α2-AR using [3H]UK 14304 (Gerhardt et al., 1990) or to β2-AR using other radiolabeled agonists (Stadel et al., 1980).
[125I]PIC binding to the D79Nα2AAR is relatively insensitive to the addition of Gpp(NH)p (Fig. 1C) (Ceresa and Limbird, 1994). In contrast, both the N422D and the D79N/N422D α2AAR possess [125I]PIC binding that is readily diminished in detectability in the presence of Gpp(NH)p (Fig. 1C). Compared with the WT α2A-AR, the findings indicate that D79 is critical in regulating α2AAR functional coupling to G proteins.
The position of the Gpp(NH)p curve reflects not only the affinity of Gpp(NH)p for the G proteins involved,but also the efficiency of G protein coupling to receptors, because Gpp(NH)p binding to the G protein (and consequent G protein conformational changes) leads to dissociation of the R-G complex, decreased receptor affinity for agonist, and thus diminished trapability of the [125I]PIC binding (Fig. 1C, schematic). Because the [125I]PIC binding detected for WT and α2A-AR mutant structures in these studies was pertussis toxin-sensitive (data not shown), the implication is that similar G proteins are involved in these interactions in the transient expression studies performed. Consequently, differences in the EC50 for Gpp(NH)p reflect differences in R-G coupling efficiency and retrograde G→R signaling from a similar pool of G proteins to different (WT versus mutant) receptor structures. Gpp(NH)p is more potent in decreasing high affinity agonist binding to the N422D [EC50 64 ± 7.5 nM (mean ± SE);n = 3] and D79N/N422D α2AAR (EC50 22 ± 4 nM; n = 3) structures when compared with wild-type α2AAR (EC50 257 ± 39 nM (mean ± SE;n = 3; Fig. 1C). A leftward shift in the Gpp(NH)p concentration response curve for decreasing [125I]PIC trapability denotes anincreased efficiency with which Gpp(NH)p dislodges high efficiency R-G interactions and is consistent with the interpretation that the functional interface between the D79N/N422D α2AAR or the N422D α2AAR may be more fragile than between the WT α2AAR and thus more easily disrupted by guanine nucleotides.
Allosteric Modulation of Ligand Binding by Na+Correlates with Membrane-Embedded Asp, but Not Asn, Residues.
Agonist (epinephrine) competition for binding of the radiolabeled antagonist ([3H]RX821002), in the presence or absence of Na+, was used to evaluate allosteric modulation of ligand binding to the α2AAR by monovalent cations (Tsai and Lefkowitz, 1978; Michel et al., 1980;Nunnari et al., 1987). As shown in Fig. 1D, the wild-type α2AAR exhibits allosteric modulation, as manifested by a rightward shift of the epinephrine competition curve in the presence of Na+ because of Na+-evoked decreases in receptor affinity for agonists. This shift in the competition curve in the presence of Na+ solely reflects a decrease in the receptor affinity for epinephrine, because [3H]RX821002 seems to be insensitive to allosteric modulation in its affinity for α2AAR (MacMillan et al., 1996) in contrast to Na+-evoked increases in receptor affinity for the antagonist [3H]yohimbine (Nunnari et al., 1987). Allosteric modulation of agonist binding by Na+ is lost in the D79N α2AAR, indicated by a lack of effect of Na+ on epinephrine competition for [3H]RX821002 (Fig. 1E), corroborating the role of aspartate D79 in allosteric modulation of ligand binding by cations in the α2AAR (Horstman et al., 1990) and in a variety of other GPCR (Neve et al., 1991; Kong et al., 1993; Tian and Deth, 1993). Interchange of the aspartate in TM7 with the asparagine in TM7, creating the D79N/N422D α2AAR double mutant, creates an intermediate phenotype of allosteric modulation of ligand binding (Fig. 1G). Not surprisingly, the single mutant N422D α2AAR (Fig. 1F) also exhibits allosteric modulation of agonist binding by Na+, because this structure contains an aspartate in both TM2 and TM7 that both could serve as negative counterions for the Na+cation. All of the α2AAR mutants studied (i.e., D79N, N422D, or D79N/N422D) seem to possess a higher affinity for agonist than the wild-type α2AAR, manifested as a shift to the left in the agonist competition curve in the absence of Na+ [receptor structure EC50 (n = 3): WT, 9.5 ± 2 μM; D79N, 1.4 ±1 μM*; N422D, 1.0 ± 0.5 μM*; D79N/N422D, 1.6 ± 0.8 μM* (*p < 0.05 compared with WT) (Fig. 1, D-G)], as reported previously for the D79N mutation in the α2AAR (Horstman et al., 1990; Lakhlani et al., 1997).
Impact of the Presumed α2AAR Asp79(TM2)/Asn422(TM7) Interface on MAP Kinase Activation.
Previous studies have demonstrated a selective inability of the D79Nα2AAR to activate G protein βγ-dependent pathways (Surprenant et al., 1992), such as the activation of receptor-operated K+ currents (Clapham and Neer, 1993) compared with α subunit-involved pathways such as inhibition adenylyl cyclase or voltage-gated Ca2+ currents (Surprenant et al., 1992; Lakhlani et al., 1996). Because the D79N mutation is also known to result in ablation of allosteric modulation of ligand binding (Horstman et al., 1990), it is reasonable to postulate a functional link between allosteric modulation of receptor conformation and activation of βγ-dependent pathways, corresponding to the postulated link between Na+ modulation of receptor affinity and receptor-βγ subunit interactions (Costa et al., 1992; Onaran et al., 1993).
GPCR-mediated activation of the MAP kinase cascade is thought to occur primarily through a βγ-dependent pathway, especially for the α2AAR (van Biesen et al., 1995). As shown in Fig. 2, the wild-type α2AAR is able to activate the MAP kinase in response to epinephrine in a concentration-dependent manner in HEK 293 cells. Activation of the MAP kinase is not detected for the D79Nα2AAR, despite similar levels of D79N and mutant receptor expression as WT receptor in these experiments. Epinephrine activates MAP kinase via the N422Dα2AAR, although with a trend of a reduced potency (note trend of rightward shift in the EC50 value for epinephrine to activate MAP kinase in N422D α2A-AR-expressing cells compared with cells expressing the WTα2A-AR) contrasting with the increased affinity for agonist ligand for this receptor structure (Fig. 1, D and G). Taken together, these observations indicate that the efficacy of N422Dα2AAR coupling to G proteins is less than that of the wild-type α2AAR, corroborating the interpretations of the increased sensitivity of125I-PIC binding to Gpp(NH)p for the N422Dα2AAR (Fig. 1C). Interchange of residues 79 and 422, creating the D79N/N422D double mutant α2AAR, also permits MAP kinase activation by epinephrine in a dose-dependent manner, but with a diminished potency compared with the wild-type α2AAR (Fig. 2 and Tables 1 and2). This finding is again consistent with the interpretation of findings in Fig. 1 that the D79N and N422D residues contribute to an interface between TM2 and TM7 that regulates receptor signaling properties.
Mutation of Residues 79 or 422 Alters Cell Surface Receptor Turnover and Results in Ligand Modulation of Receptor Turnover and Density.
Recently, we demonstrated that the D79N α2AAR manifests a higher rate of cell-surface turnover and that this turnover can be slowed by receptor structural stabilization with either agonist or antagonist occupancy, resulting in steady-state receptor density up-regulation (Wilson and Limbird, 2000). The N422Dα2AAR, despite retention of allosteric modulation of ligand binding by Na+(Fig. 2I), possesses a surface t 1/2 of <3 h compared with the 13 ± 1.0 h surfacet 1/2 of wild-type α2AAR (Fig. 3A). The N422Dα2AAR also exhibits receptor density up-regulation by antagonist but not by agonist (Fig. 3C); similarly, antagonist, but not agonist, occupancy of the receptor slows cell surface receptor turnover (Fig. 3B).
Interchange of residues 79 and 422 (D79N/N422D) of the α2AAR slows the surfacet 1/2 to 4.7 ± 0.3 h for the D79N/N422D α2AAR (Fig. 3A) compared with the <3 h t 1/2 characteristic of the single mutant N422D α2AAR. Turnover of the D79N/N422Dα2AAR is slowed by occupancy of the mutant α2AAR with either agonist or antagonist (Fig. 3B) and both agonist and antagonist similarly up-regulate receptor density of the D79N/N422Dα2AAR (Fig.3C), recapitulating findings for the D79Nα2AAR (Fig. 3, B and C). Thus, although the D79N/N422D double mutant exhibits allosteric modulation by Na+ and functional α2AAR-G protein coupling (Fig. 1), receptor stabilization on the cell surface is not fully restored to that characteristic of the WT α2AAR, analogous to findings for MAP kinase activation by epinephrine at the D79N/N422D α2AAR.
Consequences of Mutations At the Presumed Asp79(TM2)/Asn422(TM7) Interface on α2aAR Conformational Stability.
Accelerated cell surface receptor turnover, in the context of ligand-mediated increases in receptor density expression in cells, can be a manifestation of the structural or conformational lability of a receptor (Wilson and Limbird, 2000). A direct measure of conformational lability in mutant GPCRs is a comparison of the rate of loss of receptor binding capability (a reflection of conformational/structural stability) with the rate of loss of receptor protein (a reflection of protein stability) in detergent-solubilized preparations (Gether et al., 1997; Wilson and Limbird, 2000). As shown in Fig.4, the N422D α2AAR is extremely structurally/conformationally unstable compared with wild-type or even D79N α2AAR (Fig. 4), which might be expected given the presumed juxtaposition of two negative charges within the bilayer of the α2AAR structure. Placing the D79N mutation in the N422D structure (D79N/N422D) creates an α2AAR structure with an intermediate phenotype for conformational stability compared with either single mutation (D79N or N422D) alone (Fig. 4), based on the approximatet 1/2 of functional binding capacity in detergent solution of 10 min for N422D, 1.5 h for D79N and 20 min for D79N/N422D.
Additional Mutagenesis at Residue 79 Reveals the Critical Importance of the Aspartate 79 Residue Per Se in Regulating Receptor Cell-Surface Residence Time and Structural Stability.
The observation that interchanging the aspartate in TM2 with the asparagine in TM7, generating the D79N/N422D α2AAR double mutant, diminishes cell-surface residence time and enhances structural lability in detergent solution when compare to the wild-type α2AAR implies that simply complementing the asparagine at 79 with a negative charge at this TM2/TM7 interface (N422D) is not sufficient to impart functional properties identical with that of the wild-type receptor. To explore this interpretation further, two additional mutant α2AAR structures were examined. The D79E α2AAR substitutes the negatively charged aspartate with a negatively charged glutamate at this TM2/TM7 interface. Previous studies from our laboratory have demonstrated that the D79E α2AAR couples to G proteins and is allosterically modulated by cations in a manner indistinguishable from the WT α2AAR (Ceresa and Limbird, 1994). However, mutation of aspartate to glutamate does more than simply substitute a negative charge with another, but also extends the presumed negative charge by one methylene group. Another mutation of residue 79, the D79Q α2AAR, previously demonstrated to diminish G protein coupling efficiency and eliminate allosteric modulation by cations (Ceresa and Limbird, 1994), also was examined. The rate of cell-surface turnover of both the D79E and D79Q α2AAR is more rapid than that of the wild-type α2AAR (Fig. 3A). Additionally, both the D79E and D79Q α2AAR are up-regulated in receptor density after incubation with either agonist or antagonist in intact cells (Fig. 3C), presumably because of ligand-mediated receptor stabilization and slowing of cell-surface receptor turnover (Fig. 3B).
Analysis of the structural stability of the D79E and D79Q α2AARs reveals that even the presumed conservative substitution of aspartate 79 with glutamate results in accelerated loss of binding activity in detergent compared with wild-type α2AAR (Fig. 4). Interestingly, the rate of loss of binding capability in detergent for the D79E α2AAR parallels the rate of loss for the D79N/N422D α2AAR. These are important findings, because they indicate that allosteric modulation by cations and coupling to G proteins, both of which are true for the D79E and D79N/N422D α2AARs, do not necessarily rely on or predict a conformationally/structurally stable receptor structure [(Ceresa and Limbird, 1994) and Figs. 1-3].
Discussion
Cell-surface receptor expression is necessary for surface-mediated GPCR signaling. Conformational stability contributes to maintaining cell surface receptor expression of GPCR in general and the α2AAR in particular. Mutant α2-AR receptors lacking conformational stability exhibit a faster removal from the cell surface that can be slowed by receptor occupancy with ligand [Fig. 3 and 4 and (Wilson and Limbird, 2000)]. Previous investigations have focused on ligand mediated up-regulation of mutant GPCRs through either increased cell-surface delivery (Morello et al., 2000) or increased cell-surface residence time (Wilson and Limbird, 2000). The present study suggests that the conserved D79(TM2)/N422(TM7) interface contributes to conformational stability of the α2AAR, and, because of the conserved nature of this interface, such findings may be generalizable to other GPCRs. The multiple functional and structural properties evaluated in this study for mutations at this interface are summarized in Tables 1 and 2.
Considerable modeling of the predicted interactions among amino acid side chains in the TM spans of GPCR has been undertaken, particularly for receptors that bind monoamines (Sealfon et al., 1995; Mizobe et al., 1996; Gether and Kobilka, 1998). These models, developed to describe a molecular basis for experimental data (Zhou et al., 1994;Sealfon et al., 1995), suggest that TM2 and TM7 are in near each other and that a hydrogen bonding network, including an aspartate in TM2 (D79 of the α2AAR) and an asparagine in TM 7 (N422 of the α2AAR), regulates receptor activation (Fig. 1, A and B). Therefore, to interpret our results in the context of the α2A-AR, it was first important to evaluate mutations at this interface with regard to receptor activation.
Allosteric modulation of ligand binding to the α2AAR seems to be dependent on a negative charge at the TM2/TM7 interface. All receptor structures that possess a negative charge in either TM2 (wild-type), TM7 (D79N/N422D), or both TM2 and TM7 (N422D) result in allosteric modulation by monovalent cations (Fig. 1, Tables 1 and 2). The ability of receptor to be allosterically modulated by cations correlates with G protein coupling efficiency, because receptors that undergo allosteric modulation (wild-type, N422D, or D79N/N422D) also exhibit high affinity guanine nucleotide-sensitive agonist binding (Fig. 1C and Table 1). In contrast, receptors that lack allosteric modulation by cations (D79N) do not possess guanine-nucleotide sensitive agonist binding (Fig. 1, C and D; Table 1), a measure of retrograde coupling of G proteins to cognate receptors (Ceresa and Limbird, 1994). The ability of receptors to couple to G proteins in membrane preparations correlates with the ability of receptor to activate MAP kinase in intact cells, a measure of anterograde receptor to G protein coupling. Thus, receptors that undergo modulation of ligand binding by cations (wild-type, N422D, and D79N/N422D α2AARs) also activate the MAP kinase cascade; those that lack allosteric modulation by cations or regulation of agonist binding by Gpp(NH)p (e.g., the D79N α2AAR in Fig. 1, C and E) do not. Collectively, these results regarding allosteric modulation by Na+ and receptor-G protein coupling correlate with data previously obtained for other GPCR with regards to modulating the structure of GPCR at the presumed TM2/TM7 interface (Table 1).
The conserved TM2/TM7 interface also seems to play some role in regulating receptor cell surface residence time. Thus, mutation of D79 to N, E, or Q in the α2AAR is paralleled by faster cell surface receptor turnover (Fig. 3) and structural instability (Fig. 4), despite differing consequences on receptor activation (Wilson and Limbird, 2000). Mutation of TM7 at the TM2/TM7 interface to create a receptor with presumably apposing negative charges (i.e., the N422D α2AAR) leads to an extremely unstable α2AAR molecule, as measured by multiple independent lines of evidence including: 1) accelerated rate of loss functional binding capacity after detergent solubilization (Fig. 4); 2) increased cell surface receptor turnover (Fig. 3A); 3) ligand-stabilized attenuation of receptor turnover (Fig. 3B); and 4) ligand-mediated, steady-state up-regulation of receptor density (Fig.3C). It is probable that the extreme structural instability of the N422Dα2AAR results from apposition of two negatively charged residues near each other within the transmembrane domain core of the α2AAR. It is interesting that only antagonist occupancy, but not agonist occupancy, serves to stabilize the surface residence of the N422Dα2AAR, resulting in dramatic up-regulation of N422Dα2AAR density. It is possible that antagonist occupancy of the receptor stabilizes a conformation in which the interaction of the negatively-charged residues D79(TM2) and N422D(TM7) does not occur. In contrast, the agonist epinephrine, by stabilizing a distinct conformation, may foster a more direct interaction of these two residues, resulting in a considerably more unstable receptor structure, thus accounting for the lack of effect of epinephrine on cell surface receptor stabilization and resultant up-regulation of steady state receptor density. Such an interpretation is consistent with other lines of evidence that agonists and antagonists serve to stabilize differing receptor conformations (Gether and Kobilka, 1998). Placing the D79N mutation within the N422Dα2AAR structure restores some structural stability, which manifests as a decrease in the loss of functional binding capacity after detergent solubilization (Fig. 4) and a slower surface turnover in intact cells (Fig. 3A), presumably because of removal of one of the negative charges at this interface.
The properties of the D79E and D79N/N422D α2AAR structures, compared with those of wild-type α2AAR, reveal the critical importance of the precise structural localization of the negative charge on the side chain of residue 79 in regulating receptor structural stability. Although the D79E α2AAR couples to G proteins and its binding of agonist and antagonist ligands is modulated by monovalent cations in a way that is indistinguishable from the WT α2AAR (Ceresa and Limbird, 1994), the D79E α2AAR exhibits structural instability like that of the D79N/N422D α2AAR double mutant (Fig. 4). Thus, although a negative charge at residue 79 is necessary to permit functional activity of the α2AAR, it is not sufficient to afford all of the properties of the wild-type receptor, including intrinsic conformational/structural stability and prolonged receptor cell-surface residence time (Table 2).
Our data are consistent with a proximity of the TM2 and TM7 transmembrane helices, by analogy with the amine binding 5HT2A receptor and the peptide binding GnRH-R and μ-opioid receptor (Zhou et al., 1994; Sealfon et al., 1995; Flanagan et al., 1999; Xu et al., 1999). It is probable that this interface involves multiple independent or interdependent contact sites. However, our data are not necessarily consistent with a direct charge pairing of D79 in TM2 with N442 in TM7, because “swapping” of these residues via mutagenesis does not create a receptor structure (D79N/N422D) with functional or stability properties indistinguishable from the wild-type receptor. This interpretation is consistent with recent crystallographic findings for another GPCR, rhodopsin, suggesting that a molecule of water bridges these two residues (Palczewski et al., 2000). Nonetheless, our data are consistent with the interpretation that the two residues do contribute to this TM2-TM7 interface, which regulates multiple biochemical properties of the α2A-AR (Tables 1 and 2).
For the α2AAR, possessing a negative charge in TM2(D79), TM7 (D79N/N422D), or both TM2 and TM7 (N422D) is sufficient to impart allosteric modulation of ligand binding by monovalent cations, coupling to G proteins, and activation of MAP kinase activity. However, this interface also is essential for maintaining intrinsic receptor conformational/structural stability. Receptor structural stability seems to be more sensitive than receptor activation to structural modification by mutation of residues at this interface, borne out by the observation of the extreme structural instability of the N422D α2AAR as well as structural instability of the conservatively substituted D79E α2AAR. Taken together, these data suggest that this TM2/TM7 interface in GPCRs is involved in two distinct phenomena: receptor activation (measured by G protein coupling and activation of downstream effectors) and receptor conformational stability (measured directly by following receptor binding in detergent solution over time and indirectly as receptor cell-surface residence time) (Tables 1 an 2). Elucidation of sites critical for regulating or maintaining GPCR conformational/structural stability as well as cell-surface expression is an important step in providing novel therapeutic/pharmacological modulation of GPCR. This highly conserved TM2/TM7 interface seems to be one such critical site, especially because of its role in regulating receptor activation.
Acknowledgments
We thank Carol Ann Bonner for superior technical assistance with cell culture for these studies and Stephen W. Edwards, Ph.D., for helpful advice regarding the molecular modeling studies. M.H.W. would like to thank his Ph.D. dissertation committee for helpful advice and discussions. Finally, we thank all members of the Limbird lab for shared enthusiasm and support.
Footnotes
- Received October 16, 2000.
- Accepted January 3, 2001.
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Send reprint requests to: Dr. Lee E. Limbird, Department of Pharmacology, 468 MRB I, Vanderbilt University Medical Center, Nashville, Tennessee. E-mail:lee.limbird{at}mcmail.vanderbilt.edu
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This work was supported by National Institutes of Health Grant HL43671 to L.E.L. and Medical Scientist Training Program Grant GM07347 for M.H.W.
Abbreviations
- α2AR
- α2-adrenergic receptor
- GPCR
- G protein-coupled receptors(s)
- TM
- transmembrane domain
- MAP
- mitogen activated protein
- GnRH
- gonadotropin-releasing hormone
- HA
- hemagglutinin
- CHO
- Chinese hamster ovary
- EcR
- ecdysone receptor-expressing
- HEK
- human embryonic kidney
- FCS
- fetal calf serum
- PMSF
- phenylmethylsulfonyl fluoride
- [125I]PIC
- para-[125I]iodoclonidine
- GppNHp
- 5′-guanylylimidodiphosphate
- WT
- wild-type
- PAGE
- polyacrylamide gel electrophoresis
- DβM/CHS
- dodecyl-β-d-maltoside/cholesteryl hemisuccinate
- CAPS
- 3-(cyclohexylamino)propanesulfonic acid
- t1/2
- half-life
- The American Society for Pharmacology and Experimental Therapeutics