An asn to lys polymorphism in the third intracellular loop of the human alpha 2A-adrenergic receptor imparts enhanced agonist-promoted Gi coupling.

alpha(2A)-Adrenergic receptors (alpha(2A)AR) are presynaptic autoinhibitory receptors of noradrenergic neurons in the central and peripheral sympathetic nervous systems, which act to dynamically regulate neurotransmitter release. Signaling through the G(i)/G(o) family of G-proteins, the receptor subserves numerous homeostatic and central nervous system functions. A single nucleotide polymorphism of this receptor, which results in an Asn to Lys substitution at amino acid 251 of the third intracellular loop, was identified in the human population. The frequency of Lys-251 was 10-fold greater in African-Americans than in Caucasians, but was not associated with essential hypertension. To determine the consequences of this substitution, wild-type and Lys-251 receptors were expressed in CHO and COS-7 cells. Expression, ligand binding, and basal receptor function were unaffected by the substitution. However, agonist-promoted [(35)S]GTPgammaS binding was approximately 40% greater with the Lys-251 receptor. This enhanced agonist function was observed with catecholamines, azepines, and imidazolines albeit to different degrees. In studies of agonist-promoted functional coupling to G(i), the polymorphic receptor displayed enhanced inhibition of adenylyl cyclase (60 +/- 4. 4 versus 46 +/- 4.1% inhibition) and markedly enhanced stimulation of MAP kinase (57 +/- 9 versus 15- +/- 2-fold increase over basal) compared with wild-type alpha(2A)AR. The potency of epinephrine in stimulating inositol phosphate accumulation was increased approximately 4 fold with the Lys-251 receptor. Unlike previously described variants of G-protein-coupled receptors, where the minor species causes either a loss of function or increased non-agonist function, Lys-251 alpha(2A)AR represents a new class of polymorphism whose phenotype is a gain of agonist-promoted function.


MATERIALS AND METHODS
Polymorphism Detection-The intronless human ␣ 2A AR gene (Gen-Bank accession number AF281308, which includes the sequence corrections illuminated by Guyer (13)) was amplified by overlapping PCR reactions from genomic DNA derived from blood samples. The 1350-bp coding sequence as well as 341-bp 5Ј-UTR and 174-bp 3Ј-UTR were examined. In this paper the adenine of the initiator ATG codon is designated as nucleotide 1, and amino acid 1 is the encoded methionine. The human receptor consists of 450 amino acids. For initial examination, DNA from 27 hypertensive individuals was utilized. Overlapping PCR products encompassing the ␣ 2A gene were designated fragments 1-5 and were generated using the following primers: Fragment 1 (600 bp), 5Ј-TTTACCCATCGGCTCTCCCTAC-3Ј (sense) and 5Ј-GAGACAC-CAGGAAGAGGTTTTGG-3Ј (antisense); Fragment 2 (467 bp), 5Ј-TCGTCATCATCGCCGTGTTC-3Ј (sense) and 5Ј-CGTACCACTTCTGG-TCGTTGATC-3Ј (antisense); Fragment 3 (556 bp), 5Ј-GCCATCATCA-TCACCGTGTGGGTC-3Ј (sense) and 5Ј-GGCTCGCTCGGGCCTTGCC-TTTG-3Ј (antisense); Fragment 4 (436 bp), 5Ј-GACCTGGAGGAGAGC-TCGTCTT-3Ј (sense) and 5Ј-TGACCGGGTTCAACGAGCTGTTG-3Ј (antisense); and Fragment 5 (353 bp), 5Ј-GCCACGCACGCTCTTCAA-ATTCT-3Ј (sense) and 5Ј-TTCCCTTGTAGGAGCAGCAGAC-3Ј (antisense). The 5Ј-end of each sense and antisense primer also contained sequence corresponding to the M13 Forward (5Ј-TGTAAAACGACGG-CCAGT) and M13 Reverse (5Ј-CAGGAAACAGCTATGACC) universal sequencing primers, respectively. The PCR consisted of ϳ100 ng of genomic DNA, 5 pmol of each M13 primer, 0.8 mM dNTPs, 10% dimethyl sulfoxide, 2.5 units of Platinum taq DNA polymerase (Life Technologies, Inc.), 20 l of 5ϫ buffer A (Invitrogen) in a 100-l reaction volume. Reactions were started by an initial incubation at 94°C for 4 min, followed by 35 cycles of 94°C for 30 s, denaturation for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 7 min. The denaturation temperature was 56°C for fragments 1 and 5, 58°C for fragments 2 and 4, and 60°C for fragment 3. PCR reactions were purified using the QIAquick PCR purification system (Qiagen), and automated sequencing of both strands of each PCR product was performed using an Applied Biosystems sequencer using dye primer methods. As discussed below, a C to G transversion at nucleotide 753 was identified that resulted in an asparagine to lysine change at amino acid 251 (see Fig. 1). This nucleotide change resulted in gain of a unique StyI restriction endonuclease site in PCR fragment 3, and the presence or absence of this polymorphism in additional samples was studied by StyI digestion of fragment 3 PCR products (see Fig. 1D). This rapid detection technique was applied to additional DNA samples providing genotypes at this locus from a total of 376 individuals (normotensive: 125 Caucasian and 99 African-American; hypertensive: 75 and 77, respectively). Normotensive and hypertensive patients were selected as described previously (14).
Constructs and Cell Transfection-To create the polymorphic ␣ 2A Lys-251 construct, a portion of the coding region of the ␣ 2A AR gene containing a G at nucleotide position 753 was amplified from a homozygous individual using fragment 2 sense and fragment 4 antisense primers (see PCR conditions described above). This fragment was digested with and subcloned into the BglII and SacII sites of the wild-type ␣ 2A AR sequence in the expression vector pBC12B1. Chinese hamster ovary cells (CHO-K1) were permanently transfected by a calcium phosphate precipitation technique as described previously using 30 g of each receptor construct and 3.0 g of pSV 2 neo to provide for G418 resistance (15). Selection of positive clones was carried out in 1.0 mg/ml G418, and expression of the ␣ 2A AR from individual clonal lines was determined by radioligand binding as described below. Cells were grown in monolayers in Ham's F-12 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 80 g/ml G418 (to maintain selection pressure) at 37°C in a 5% CO 2 atmosphere. COS-7 cells were co-transfected with 1-10 g of each ␣ 2A AR construct and 5 g of G i␣2 using a DEAE-dextran method essentially as described previously (16). These transfections also included 5 g of the large T antigencontaining plasmid, pRSVT (17), to maximize expression of the ␣ 2A AR gene from the SV40 promoter of pBC12B1.
Adenylyl Cyclase Activities-␣ 2 AR inhibition of adenylyl cyclase was determined in membrane preparations from CHO cells stably expressing the two receptors using methods similar to those previously described (18). Reactions consisted of 20-g cell membranes, 2.7 mM phosphoenolpyruvate, 50 M GTP, 0.1 mM cAMP, 0.12 mM ATP, 50 g/ml myokinase, 0.05 mM ascorbic acid, and 2 Ci of [␣- 32   MAP Kinase Activation-Activation of p44/42 MAP kinase was determined by quantitative immunoblotting using specific antibodies to identify phosphorylated and total MAP kinase expression. Briefly, confluent cells were incubated overnight in serum-free media prior to treatment with media alone (basal), epinephrine (10 M), or thrombin (1 unit/ml) for 5 min. Cells were washed three times with phosphatebuffered saline (PBS) then lysed in RIPA (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 5 mM NaF) containing protease inhibitors (10 g/ml benzamidine, 10 g/ml soybean trypsin inhibitor, 10 g/ml aprotinin, and 5 g/ml leu-peptin). Western blots of these whole cell lysates were performed essentially as described previously (19). Membranes were incubated with phospho-p44/42 MAP kinase E10 antibody (New England BioLabs, Beverly, MA) at a dilution of 1:2000 for 1 h at room temperature. Washed membranes were subsequently incubated with anti-mouse fluorescein-linked immunoglobulin followed by incubation with fluorescein alkaline phosphatase (ECF, Amersham Pharmacia Biotech). Fluorescent signals were quantitated by real-time acquisition using a Molecular Dynamics Storm imager. After stripping, membranes were incubated under the same conditions as above with a p44/42 MAP kinase monoclonal antibody to quantitate total MAP kinase expression.
Inositol Phosphate Accumulation-Total inositol phosphate levels in intact cells were determined essentially as described previously (20). Briefly, confluent CHO cells stably expressing each of the ␣ 2A ARs were incubated with [ 3 H]myoinositol (5 Ci/ml) in media lacking fetal calf serum for 16 h at 37°C in 5% CO 2 atmosphere. Subsequently, cells were washed and incubated with PBS for 30 min followed by a 30-min incubation with 20 mM LiCl in PBS. Cells were then treated with PBS alone (basal), varying concentrations of epinephrine, or 5 units/ml thrombin for 5 min, and inositol phosphates were extracted as described by Martin (21). Following separation on Agl-X8 columns, total inositol phosphates were eluted with a solution containing 0.1 M formic acid and 1 M formate.
Radioligand Binding-Expression of mutant and wild-type ␣ 2A AR  was determined using saturation binding assays as described (22)  Miscellaneous-Protein determinations were by the copper bicinchoninic acid method (23). Data from adenylyl cyclase and radioligand binding assays were analyzed by iterative least-square techniques using Prizm software (GraphPad, San Diego, CA). Agreement between genotypes observed and those predicted by the Hardy-Weinberg equilibrium was assessed by a Chi-squared test with one degree of freedom. Genotype comparisons were made using Fisher's exact test. Comparisons of results from biochemical studies were by paired t tests, and significance was considered when p Ͻ 0.05. Data are provided as means Ϯ S.E.

RESULTS AND DISCUSSION
Sequence analysis of the entire coding region of the ␣ 2A AR gene from 54 chromosomes revealed one nonsynonymous sequence variant located within the third intracellular loop of the receptor (Fig. 1). This consisted of a C to G transversion at nucleotide 753 that changed amino acid 251 from Asn to Lys (Fig. 2). Although the Lys-251 receptor is relatively rare, it is ϳ10-fold more common in African-Americans than in Caucasians, with an allele frequency of 0.05 as compared with 0.004 (p ϭ 0.01). The distribution of homozygous and heterozygous alleles was not different than that predicted from Hardy-Weinberg equilibrium (p Ͼ 0.9). Two previously unreported synonymous single nucleotide polymorphisms were also identified at nucleic acids 849 (C to G) and 1093 (C to A). Considering the role of the ␣ 2A AR in regulating blood pressure, we also determined the frequency of this polymorphism in patients with essential hypertension. Our analysis of 99 normotensive and 77 hypertensive African-Americans as well as 125 normotensive and 75 hypertensive Caucasians showed no differences in the frequency of this polymorphism in patients with essential hypertension in either group.
The consequences of this polymorphism on ligand binding and receptor function were evaluated by permanently expressing the human wild-type ␣ 2A AR and the Lys-251 polymorphic receptor in CHO cells. Saturation radioligand binding studies revealed essentially identical dissociation binding constants for the ␣ 2 AR antagonist [ 3 H]yohimbine (K d ϭ 3.4 Ϯ 0.21 versus 3.6 Ϯ 0.25 nM, respectively, n ϭ 4), and competition binding assays showed no differences in binding of the agonist (Ϫ) epinephrine (K i ϭ 593 Ϯ 65 versus 734 Ϯ 31 nM respectively, n ϭ 3, Table I). These data confirm that the ligand binding pocket composed of the transmembrane spanning domains is not perturbed by the presence of Lys at amino acid 251 in the third intracellular loop.
The Lys-251 polymorphism occurs in a highly conserved portion of the third intracellular loop of the ␣ 2A AR (Fig. 2), in a region thought to be important for G-protein interaction (24). Indeed, as shown in Fig. 2, Asn is present in the position analogous to human 251 in all mammalian ␣ 2A ARs reported to date. To assess whether this polymorphism affects G-protein coupling, functional studies examining agonist-promoted inhibition of forskolin-stimulated adenylyl cyclase activities were carried out in cell lines expressing the wild-type Asn-251 receptor and the polymorphic Lys-251 receptor at levels of 2360 Ϯ 263 and 2590 Ϯ 140 fmol/mg (n ϭ 5, p Ͼ 0.05), respectively. As shown in Table I, basal and 5.0 M forskolin-stimulated adenylyl cyclase activities were not different between Asn-251-and Lys-251-expressing cell lines, indicating that non-agonist-dependent function is equivalent with the two receptors. However, activation of the polymorphic Lys-251 receptor with the full agonist epinephrine resulted in enhanced inhibition of adenylyl cyclase activity compared with wild-type ␣ 2A AR. Maximal inhibition of adenylyl cyclase was 60 Ϯ 4.4% with the variant receptor compared with 46 Ϯ 4.1% with wild-type (n ϭ 5, p Ͻ 0.005, Fig. 3A). Similar results were also found when receptors were activated by the partial agonist oxymetazoline, with the Lys-251 having an ϳ40% augmented function compared with the Asn-251 receptor (50 Ϯ 6.6% versus 35 Ϯ 4.7% inhibition, n ϭ 5, p Ͻ 0.05, Fig. 3B transfected G i , epinephrine promoted [ 35 S]GTP␥S binding was only 12 Ϯ 2.6% over basal, compared with 57 Ϯ 11% over basal when G i␣2 was co-expressed. So, these studies primarily quantitate ␣ 2A AR-G i␣2 coupling as opposed to G-proteins, which are endogenously expressed in these cells. As is shown in Fig. 4, the Lys-251 receptor displayed increased [ 35 S]GTP␥S binding in response to all agonists tested, albeit to varying degrees. Basal [ 35 S]GTP␥S binding was equivalent. Stimulation with the full agonists UK-14304, epinephrine, and norepinephrine resulted in ϳ40% enhanced GTP␥S binding for the Lys-251 receptor as compared with the Asn-251 receptor. On the other hand, partial agonists displayed from 45% (BHT-933) up to 72% (guanabenz) enhancement of [ 35 S]GTP␥S binding with the Lys-251 receptor. These results are consistent with the adenylyl cyclase activity studies, which also showed enhanced function of the polymorphic receptor. In addition, they indicate that the gainof-function phenotype is more pronounced with some, but not all, partial agonists compared with full agonists.
We next investigated agonist-mediated modulation of MAP kinase by wild-type and the Lys-251 receptor. ␣ 2 AR act to stimulate MAP kinase activity and thus can potentially regulate cell growth and differentiation (2). Although noting that regulation of MAP kinase activity is both receptor and cell-type specific, MAP kinase activation by ␣ 2A AR receptors appears to be initiated by ␤␥ released from G i (2). To investigate the extent of MAP kinase activation in CHO cell lines expressing both the Asn-251 and Lys-251 receptors, quantitative immunoblots using an antibody specific to the activated (phosphorylated) form of ERK 1/2 were performed. Although the total amount of MAP kinase was not different (Fig. 5A), agonist-promoted stimulation of MAP kinase activity was markedly different between the two cell lines (Fig. 5, A and B). Activation of the Lys-251 receptor with 10 M epinephrine resulted in a 57 Ϯ 9-fold increase in MAP kinase activity over basal as compared with 15 Ϯ 2.1-fold increase with the Asn-251 receptor (n ϭ 3, p ϭ Ͻ0.05).
Finally, coupling of these two receptors to the stimulation of inositol phosphate production was examined. Such ␣ 2 AR signaling is a complex response due to activation of phospholipase C by ␤␥ released from activated G o and G i (25). Maximal epinephrine-stimulated accumulation of inositol phosphates was not found to be different between Lys-251-and Asn-251expressing cells (Fig. 6). However, the EC 50 was lower with the Lys-251 receptor (119 Ϯ 36 nM) than the wild-type ␣ 2A AR (493 Ϯ 21 nM, p Ͻ 0.05). Thus signaling to PLC is also enhanced with the polymorphic receptor based on this increase in agonist potency.
␣ 2A ARs are widely expressed throughout the nervous system and peripheral tissues. Recent work with relatively selective agonists and antagonists, radiolabels, and specific molecular probes in several species, including genetically engineered mice, have begun to elucidate specific functions for the various ␣ 2 AR subtypes (26). The latter studies have been particularly useful in identifying subtype-specific functions. Mice lacking ␣ 2A AR have higher resting systolic blood pressures and more rapidly develop hypertension with sodium loading after subtotal nephrectomy than wild-type mice (27). Furthermore, these ␣ 2A AR knock-out mice fail to display a hypotensive response to the agonist dexmedetomidine (5). Heart rates in these mice were increased at rest, which correlated with increased [ 3 H]norepinephrine release from cardiac sympathetic nerves. These data thus indicate 1) that the presynaptic inhibition of neurotransmitter release in cortical and cardiac nerves serves important homeostatic functions in blood pressure and cardiac function, and 2) that the physiologic effects of therapeutic agonists such as clonidine reduce blood pressure by specifically acting at the ␣ 2A AR subtype. The lack of a hypotensive effect of ␣ 2 AR agonists has also been shown in genetically altered (hitand-run) mice expressing a dysfunctional ␣ 2A AR (D79N) (28). These mice also responded poorly to ␣ 2 AR agonists for several other physiologic functions (6). Dexmedetomidine failed to reduce rotarod latency or induce prolongation of sleep time, to enhance the efficacy of halothane, or to attenuate thermally induced pain in these mice. Thus the sedative, anestheticsparing, and analgesic effects of ␣ 2 AR agonists are due to activation of the ␣ 2A AR subtype. Indeed, these physiologic defects correlated with absent ␣ 2A AR regulation of inwardly rectifying K ϩ channels of locus ceruleus neurons and voltagegated Ca 2ϩ channels of these same neurons, as well as those of the superior cervical ganglion (6).
The above studies indicate that a polymorphism resulting in a markedly depressed ␣ 2A AR function in humans would likely be of physiologic importance. Indeed, such a polymorphism might well be a significant risk factor for hypertension. How- FIG. 5. Stimulation of MAP kinase by wild-type and Lys-251 ␣ 2A ARs. Phosphorylation of MAP kinase was determined in CHO cells by quantitative immunoblotting with enhanced chemifluorescence using antibodies specific for phosphorylated Erk1/2. The same blots were stripped and reprobed for total MAP kinase expression, which was not significantly different between the two cell lines (A). Cells were studied after incubation with carrier (basal), 10 M epinephrine, or 1 unit/ml thrombin. Results are shown as the -fold stimulation over basal levels (B). *, p Ͻ 0.05 compared with the wild-type response (n ϭ 3 experiments).
FIG. 6. Stimulation of inositol phosphate accumulation by wild-type and Lys-251 ␣ 2A ARs. Total inositol phosphate production in intact CHO cells was measured as described under "Materials and Methods" in response to a 5-min exposure to epinephrine. The maximal response was not different, but the EC 50 was decreased with Lys-251 compared with wild-type ␣ 2A AR (see text). Results are from five experiments. ever, the one coding block polymorphism that we found in Caucasians and African-Americans is not associated with essential hypertension and in fact its phenotype is a gain of function. It should be noted that with our sample size we have the power to detect a polymorphism with an allele frequency of 0.04 with a statistical certainty of 90%, thus it is unlikely that we have failed to detect another polymorphism that is common in any of the cohorts. Based on the phenotype of the Lys-251 receptor, and the known physiologic function of the ␣ 2A AR, one might predict that the polymorphism would predispose to autonomic dysfunction characterized by hypotension and bradycardia. Similarly, patients with essential hypertension who have the polymorphism may have milder disease or display enhanced efficacy of antihypertensive agents such as clonidine or guanabenz. Interestingly, these individuals may display more pronounced central nervous system side-effects from these agents, such as sedation, which could ultimately limit their therapeutic utility. Finally, the hyperfunctioning polymorphism would be predicted to result in less norepinephrine release from cardiac sympathetic nerves, thereby potentially providing protection against the deleterious effects of catecholamines in patients with heart failure. These scenarios await explicit testing.
Mutations of G-protein-coupled receptors are the basis of a number of rare diseases (29). In contrast, polymorphisms (allele frequencies Ͼ1%) of these receptors have been identified, which can be minor risk factors for complex diseases (30), but more importantly act as disease modifiers (31) or alter response to therapeutic agents targeting the receptor (32). Interestingly, when such mutations or polymorphisms have been found to alter function, the minor allelic variant (i.e. the least common form of the receptor) results in either decreased agonist-promoted function or increased non-agonist-dependent function (i.e. constitutive activation). An example of the former is the Ile-164 polymorphisms of the ␤ 2 AR, which imparts defective agonist-promoted coupling to G s (33). Constitutive activation results in receptors adopting a mutation-induced agonistbound like state and thus signaling becomes independent of agonist. Such persistent activation is the pathologic basis for diseases such as male precocious puberty, which is due to a mutation in the leutinizing hormone receptor (34). In the current report we show that the Lys-251 receptor does not exhibit constitutive activation, based on wild-type [ 3 S]GTP␥S binding, adenylyl cyclase, and MAP kinase activities in the absence of agonist. Instead, the phenotype that we observed was one of increased agonist-promoted function. To our knowledge this is the first delineation of a polymorphism of a pharmacogenetic locus of any G-protein-coupled receptor where the minor allele displays this property, and thus this represents a new class of polymorphism for the superfamily.