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J. Biol. Chem., Vol. 275, Issue 30, 23059-23064, July 28, 2000

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A Four Amino Acid Deletion Polymorphism in the Third Intracellular Loop of the Human _2C-Adrenergic Receptor Confers Impaired Coupling to Multiple Effectors^*

Kersten M. Small, Susan L. Forbes, Fahema F. Rahman, Kari M. Bridges, and Stephen B. Liggett

From the Departments of Medicine and Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Received for publication, February 2, 2000, and in revised form, April 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The ₂-adrenergic receptors (₂ARs) play a critical role in modulating neurotransmitter release in the central and peripheral sympathetic nervous systems. A polymorphism of the ₂AR subtype localized to human chromosome 4 (the pharmacologic _2CAR subtype) within an intracellular domain has been identified in normal individuals. The polymorphism (denoted Del322-325) is because of an in-frame 12-nucleic acid deletion encoding a receptor lacking Gly-Ala-Gly-Pro in the third intracellular loop. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were permanently transfected with constructs encoding wild-type human _2CAR and the polymorphic receptor. The Del322-325 variant had decreased high affinity agonist binding (K_H = 7.3 ± 0.95 versus 3.7 ± 0.43 nM; %R_H = 31 ± 4 versus 49 ± 4) compared with wild-type indicating impaired formation of the agonist-receptor-G protein complex. The polymorphic receptor displayed markedly depressed epinephrine-promoted coupling to G_i, inhibiting adenylyl cyclase by 10 ± 4.3% compared with 73 ± 2.4% for wild-type _2CAR. This also was so for the endogenous ligand norepinephrine and full and partial synthetic agonists. Depressed agonist-promoted coupling to the stimulation of MAP kinase (~71% impaired) and inositol phosphate production (~60% impaired) was also found with the polymorphic receptor. The Del322-325 receptor was ~10 times more frequent in African-Americans compared with Caucasians (allele frequencies 0.381 versus 0.040). Given this significant loss of function phenotype in several signal transduction cascades and the skewed ethnic prevalence, Del322-325 represents a pharmacoethnogenetic locus and may also be the basis for interindividual variation in cardiovascular or central nervous system pathophysiology.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The ₂-adrenergic receptors (₂AR)¹ are cell surface receptors for catecholamines, which couple to the G_i/G_o family of G proteins. ₂AR are expressed at multiple sites within the central and peripheral sympathetic nervous systems and may also be expressed at noninnervated sites of peripheral tissues as well. In the central nervous system presynaptic ₂AR act to inhibit the release of neurotransmitters such as norepinephrine, serotonin, and dopamine. As such, a number of responses have been ascribed to activation of these receptors by endogenous catecholamines or exogenously administered agonists. These include modulation of blood pressure, sedation, analgesia, opiate withdrawal, and multiple complex cognitive and behavioral parameters (1-5).

Three human ₂AR subtypes have been cloned and characterized and are denoted as the _2A, _2B, and _2C subtypes (6-8). Based on chromosomal localization, these have previously been denoted as ₂C10, ₂C2, and ₂C4, respectively. Recent studies including those with genetically engineered mice have shown that the _2C subtype plays specific roles in modulation of the acoustic startle reflex, prepulse inhibition, isolation-induced aggression, spatial working memory, development of behavioral despair, body temperature regulation, dopamine and serotonin metabolism, presynaptic control of neurotransmitter release from cardiac sympathetic nerves and central neurons, and postjunctional regulation of vascular tone (2-5, 9-11). The therapeutic utility of ₂AR agonists and antagonists has been limited by the lack of highly subtype-specific compounds as well as marked interindividual variability in efficacy and adverse side effects of available agents.

Given the above, and our recent delineation of functionally significant polymorphisms of the ₁- and ₂-adrenergic receptors (12-14), we have examined the genomic cDNA sequence of the _2CAR in a cohort of normal individuals. A four-amino acid deletion in the third intracellular loop was found, which was much more common in African-Americans as compared with Caucasians. Recombinant studies revealed that the deletion receptor has a distinct phenotype with a significant loss of signaling to several effector systems.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Polymorphism Detection-- The sequence encoding the third intracellular loop of the human _2C receptor (GenBank^TM assession no. J03853) was examined for polymorphic variation by performing polymerase chain reactions (PCR) to amplify this portion of the cDNA from genomic DNA derived from blood samples. 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 462 amino acids. For initial examination, DNA from 20 normal individuals was utilized. Primers for PCR were: 5'-CCACCATCGTCGCCGTGTGGCTCATCT-3' (sense) and 5'-AGGCCTCGCGGCAGATGCCGTACA-3' (antisense). The PCR consisted of ~100 ng of genomic DNA, 5 pmol of each M13 primer, 0.8 mM dNTPs, 10% Me₂SO, 2.5 units Platinum taq DNA polymerase High Fidelity (Life Technologies, Inc.), 20 µl of 5× buffer E (Invitrogen) in a 100-µl reaction volume. Reactions were started by an initial incubation at 94 °C for four minutes, followed by 35 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for seven minutes. Attempts to directly sequence this product resulted in ambiguous reads, so the product was ligated into the vector PCR2.1-TOPO (Invitrogen) and TOP 10 cells were transformed. Multiple colonies from each transformation were expanded, and the subsequently isolated DNA was sequenced using an ABI 373A automated sequencer in the forward and reverse directions using dye terminator chemistry with vector T7 and M13 primers. As is discussed below, a 12-bp deletion was found in some individuals beginning at nucleotide 964 (Fig. 1A). This results in the loss of amino acids 322-325 and thus this polymorphic receptor is denoted Del322-325. This deletion results in the loss of a NciI restriction site at nucleotide 974 (forward strand), and thus a rapid detection method was devised. Smaller (384 and 372 base pair) PCR products were produced using 5'-AGCCCGACGAGAGCAGCGCA-3' as the sense primer and the aforementioned antisense primer (same reaction conditions as above), and genomic DNA derived from blood samples as the template. Within this fragment there are either five or six NciI restriction sites depending on the presence or absence of the deletion, providing for the pattern shown in Fig. 1C. This rapid detection technique was applied to additional DNA samples providing genotypes at this locus from a total of 146 individuals.

Constructs and Cell Transfection-- To create the polymorphic _2CAR construct the larger (723 bp) PCR product described above amplified from a homozygous individual was digested and subcloned into the Bpu1102 I and Eco47 III sites of the wild-type _2CAR sequence in the expression vector pBC12BI (15). The integrity of the construct was verified by sequencing. Chinese hamster ovary cells (CHO-K1) were permanently transfected by a calcium phosphate precipitation technique as described previously using 30 µg of each receptor construct and 0.5 µg of pSV₂neo to provide for G418 resistance (15). Selection of positive clones was carried out in 1.0 mg/ml G418, and expression of the _2C receptors 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 µg/ml streptomycin, and 80 µg/ml G418 (to maintain selection pressure) at 37 °C in a 5% CO₂ atmosphere.

Adenylyl Cyclase Activities-- ₂AR inhibition of adenylyl cyclase was determined in membrane preparation from CHO cells stably expressing the two receptors using methods similar to those previously described (15). Briefly, membranes (~20 µg) were incubated with 27 µM phosphoenolpyruvate, 0.6 µM GTP, 0.1 mM cAMP, 0.12 mM ATP, 50 µg/ml myokinase, 0.05 mM ascorbic acid, and 2 µCi of [-³²P]ATP in a buffer containing 40 mM HEPES, pH 7.4, 1.6 mM MgCl₂, and 0.8 mM EDTA for 30 min at 37 °C. These conditions minimize the stimulation of adenylyl cyclase, which is observed at high agonist concentrations (16, 17). Reactions were terminated by the addition of a stop solution containing excess ATP and cAMP and ~100,000 dpm of [³H]cAMP. Labeled cAMP was isolated by gravity chromatography over alumina columns with [³H]cAMP used to quantitate column recovery. Activities were measured in the presence of water (basal), 5 µM forskolin, and 5 µM forskolin with the indicated concentrations of agonists. Results are expressed as percent inhibition of forskolin-stimulated activity.

MAP Kinase Activation-- Activation of p44/42 MAP kinase was determined by quantitative immunoblotting using a phosphospecific antibody. Briefly, confluent cells were incubated overnight at 37 °C and 5% CO₂ in serum-free media prior to treatment with media alone (basal), epinephrine (10 µM), or thrombin (1 unit/ml) for 5 min. Cells were washed three times with phosphate-buffered saline and then lysed in radioimmune precipitation buffer (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 µg/ml benzamidine, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml aprotinin, and 5 µg/ml leupeptin). Western blots of these whole cell lysates were performed essentially as described previously (18) except that polyvinylidene difluoride membranes (Amersham Pharmacia Biotech) were used and incubated with phospho-p44/42 MAP kinase E10 antibody and (after stripping) with the p44/42 MAP kinase monoclonal antibody (both from New England Biolabs, Beverly, MA) at dilutions 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.

Inositol Phosphate Accumulation-- Total inositol phosphate levels in intact cells were determined essentially as described previously (19). Briefly, confluent CHO cells stably expressing each of the _2CARs were incubated with [³H]myoinositol (5 µCi/ml) in media lacking fetal calf serum for 16 h at 37 °C in 5% CO₂ atmosphere. Subsequently, cells were washed and incubated with phosphate-buffered saline for 30 min followed by a 30-min incubation with 20 mM LiCl in phosphate-buffered saline. Cells were then treated with phosphate-buffered saline alone (basal), 10 µM epinephrine, or 5 units/ml thrombin for 5 min, and inositol phosphates were extracted as described by Martin (20). 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 _2CAR was determined using saturation binding assays as described (21) with 12 concentrations (0.5-30 nM) of [³H]yohimbine and 10 µM phentolamine used to define nonspecific binding. For competition studies, membranes were incubated in 50 mM Tris-HCL, pH 7.4, 10 mM MgSO₄, 0.5 mM EDTA with 2.0 nM [³H]yohimbine and 16 concentrations of the indicated competitor in the presence of 100 µM GppNHp for 30 min at 37 °C. Reactions for the above radioligand binding studies were terminated by dilution with 4 volumes of ice cold 10 mM Tris-HCL, pH 7.4, buffer and vacuum filtration over Whatmann GF/C glass fiber filters.

Miscellaneous-- Protein determinations were by the copper bicinchoninic acid method (22). 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 was observed, and those predicted by the Hardy-Weinberg equilibrium were assessed by a ² test with one degree of freedom. Comparisons of results from biochemical studies were paired by t tests, and significance was considered when p < 0.05. Data are provided as means ± standard errors.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

From the initial sequencing of _2CAR third intracellular loop PCR products from 40 chromosomes, one nonsynonymous sequence variant was identified (Fig. 1). This consisted of an in-frame 12-nucleotide (GGGGCGGGGCCG, sense strand) deletion beginning at nucleotide 964. This results in a loss of Gly-Ala-Gly-Pro at amino acid positions 322-325 within the third intracellular loop of the receptor (Fig. 2). Other than this deletion, the remaining encoded sequence was identical to that shown in Fig. 2. The frequencies of the wild-type and the Del322-325 polymorphic _2CARs are shown in Table I. The polymorphism is rare in Caucasians with an allele frequency of 0.040. In contrast, the frequency is ~10-fold higher (0.381) in African-Americans. The distribution of homozygous and heterozygous alleles was not different than that predicted from the Hardy-Weinberg equilibrium (p > 0.8). No other nonsynonymous polymorphisms were found in the third loop sequence. However, five synonymous single nucleotide variations were found at nucleic acids 868, 871, 933, 996, and 1167. 

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Sequence variation of the human 2CAR at nucleotides 964-975. Shown are automated sequencing chromatograms (sense strand) from individuals homozygous for the WT 2CAR (A) and Del322-325 polymorphism (B). The underlined bases in A represent the nucleotides that were found to be deleted in the polymorphic sequence (arrow in B). C, agarose gel of PCR products from wild-type homozygous (384 bp), Del322-325 homozygous (372 bp), and heterozygous individuals digested with NciI. Wild-type receptor provides for the bands at the indicated molecular sizes (two products of 6 and 1 bp are not shown). The loss of one of the six NciI sites due to the polymorphism results in a unique product of 111 bp and loss of the 82- and 41-bp products. Heterozygotes have all six fragments.

View larger version (67K): [in this window] [in a new window]   Fig. 2.   Localization of the 2CAR polymorphism. Shown is the amino acid sequence and the proposed membrane topology of the fifth and sixth transmembrane-spanning domains (TMD) and the third intracellular loop. The polymorphism results in the loss of Gly-Ala-Gly-Pro at the indicated position. The third intracellular loop (ICL) is shown in a compact form for illustrative purposes and is not intended to represent known secondary structure.

View this table: [in this window] [in a new window]   Table I Frequencies of the Del322-325 2CAR polymorphism Shown are the number of individuals with each genotype out of a total of n individuals.

The consequences of this polymorphism on receptor function were evaluated by permanently expressing the human wild-type _2CAR and the Del322-325 receptor in CHO cells and examining multiple signaling pathways. As indicated, multiple clones with similar expression levels were utilized for these studies. Saturation radioligand binding studies using the ₂AR antagonist [³H]yohimbine revealed that Del322-325 had a slightly, but statistically significant, lower affinity for the radioligand compared with wild-type _2CAR (K_d = 3.8 ± 0.55 versus 2.0 ± 0.14 nM, n = 5, p = 0.03, Table II). In competition studies with the antagonist phentolamine, however, no differences in the K_i values were found between the Del322-325 polymorphism and the wild-type _2CAR (11.1 ± 1.8 versus 10.4 ± 1.2 nM, n = 5). In competition studies with the agonist epinephrine, carried out in the absence of GTP, high and low affinity binding was detected with both receptors. However, the high affinity dissociation constant, K_H, of the Del322-325 mutant was greater (i.e. lower affinity) compared with the wild-type receptor (7.3 ± 0.95 versus 3.7 ± 0.43 nM, n = 4, p = 0.01). The percentage of receptors in the high affinity state was less with the mutant receptor (%R_H = 31 ± 4 versus 49 ± 4, p = 0.01). The K_L values were not different (584 ± 71 versus 416 ± 75 nM). Taken together, this suggested that functional coupling might be depressed with the Del322-325 receptor because of impaired formation of the high affinity agonist-receptor-G_i/G_o complex.

View this table: [in this window] [in a new window]   Table II Ligand binding properties and adenylyl cyclase activities of the wild-type and Del322-325 2CAR expressed in CHO cells [3H]yohimbine binding and adenylyl cyclase assays were performed on membranes prepared from CHO cell lines stably expressing wild-type and Del322-325 2CAR as described under "Materials and Methods." Epinephrine competition binding studies were analyzed by nonlinear regression for best fit to a two-site binding model. Adenylyl cyclase activities were determined in the presence of 5.0 µM forskolin and increasing concentration of epinephrine.

Indeed, the location of the deletion in the third intracellular loop of the receptor is within 15 residues of the sequence RRGGRR. This is a motif (BBXB or BBXXB) that has been identified in a number of receptors as a G_i coupling domain (23, 24). We considered that the deletion of the two glycines or the proline in the Del322-325 receptor may induce conformational changes affecting this region or other G protein coupling domains. Functional studies examining agonist-promoted inhibition of forskolin-stimulated adenylyl cyclase activities were carried out in lines with the wild-type _2CAR and the Del322-325 receptor at expression levels of 1375 ± 141 versus 1081 ± 157 fmol/mg (n = 5, p > 0.05) and a second set of lines with lower expressions of 565 ± 69 versus 519 ± 51 fmol/mg (n = 5, p > 0.05), respectively. The results of these studies are shown in Table II and Fig. 3. As can be seen, there is a marked functional difference between the two receptors. In the higher expressing lines (Fig. 3A), wild-type _2CAR exhibited a maximal inhibitory response of 60 ± 3%. In contrast, the Del322-325 polymorphic receptor achieved a maximal inhibition of 31 ± 2% (n = 5, p < 0.001), which represents an ~50% impairment of function. Of note, the EC₅₀ values for these responses (2.6 ± 0.74 versus 1.2 ± 0.37 nM, respectively) were not different. Results from studies with the lower expressing lines revealed an even more striking phenotypic difference between the two receptors. As is shown in Fig. 3B, at these more physiologic levels of expression, agonist-promoted inhibition of adenylyl cyclase with wild-type _2CAR was 73 ± 2.4%. In marked contrast, the Del322-325 receptor exhibited very little inhibition (10 ± 4.3%, n = 5, p < 0.001). With the low expressing Del322-325 line, the EC₅₀ in some experiments could not be calculated because of the minimal response. Analysis of the composite curve of the mean data from all experiments with this line revealed an EC₅₀ of 29.6 nM. This is in contrast to 4.3 nM calculated in a like manner for the low expressing wild-type line. A similar degree of impairment was also observed with the endogenous agonist norepinephrine (Table III). Agonist-promoted functional activities of the two higher expressing receptors were also explored with full and partial synthetic ₂AR agonists with diverse structures. (Because some of these agents were weak partial agonists, only the high expressing lines could be used.) As is shown in Table III, the Del322-325 receptor has depressed agonist-promoted coupling to inhibition of adenylyl cyclase with all the agonists tested. Similar to the responses observed with epinephrine and norepinephrine, the Del322-325 receptor showed ~50% impairment in the maximal inhibition of adenylyl cyclase compared with the wild-type _2CAR for UK14304 (full agonist) as well as BHT-933, guanabenz, clonidine, and oxymetazoline (partial agonists), with no significant differences observed in the EC₅₀ values for these responses.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Coupling of wild-type and Del322-325 2CARs to the inhibition of adenylyl cyclase. Membranes from CHO cells were prepared, and adenylyl cyclase activities were determined as described under "Materials and Methods" in the presence of 5.0 µM forskolin and the indicated concentrations of epinephrine. Results are shown as the percent inhibition of forskolin-stimulated activities (for all cell lines the fold stimulation by forskolin was ~10-fold over basal levels). A, results from two cell lines expressing the wild-type and Del322-325 receptors at 1375 ± 141 and 1081 ± 157 fmol/mg. B, results from a lower levels of expression in two other cell lines with densities of 565 ± 69 and 520 ± 51 fmol/mg, respectively. Results are from five experiments. p < 0.001 for the maximal inhibition compared with wild-type.

View this table: [in this window] [in a new window]   Table III Adenylyl cyclase activities of the wild-type and Del322-325 2CAR for full and partial agonists Adenylyl cyclase assays were performed on membranes prepared from CHO cell lines expressing wild-type and Del322-325 2CAR at 1570 ± 79.9 and 1520 ± 27.6 fmol/mg, respectively, as described under "Materials and Methods."

We next explored coupling of these two receptors to the stimulation of inositol phosphate production. In CHO cells this response is ablated by pertussis toxin, indicating coupling via G_i and/or G_o (25). The activation of phopholipase C is likely because of both G_o- and G_i-associated G stimulation of the enzyme (25). As shown in Fig. 4, the loss of function phenotype of the Del322-325 receptor as delineated in adenylyl cyclase experiments was also observed in these inositol phosphate accumulation studies. Epinephrine-stimulated accumulation of inositol phosphates was 30 ± 3% over basal with the wild-type _2CAR, compared with 11 ± 2% for the Del322-325 receptor (n = 4, p < 0.005), which amounts to an ~60% impairment of function for the polymorphic receptor. Expression levels for the two receptors for these experiments were 806 ± 140 and 733 ± 113, respectively.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Stimulation of inositol phosphate accumulation by wild-type and Del322-325 2CARs. Total inositol phosphate production in intact CHO cells was measured as described under "Materials and Methods" in response to a 5-min exposure to 10 µM epinephrine. Receptor expression was 806 ± 140 and 733 ± 113 fmol/mg, respectively, for these experiments. *, p < 0.005 compared with wild-type response (n = four experiments). IP, inositol phosphate.

Finally, agonist-mediated stimulation of MAP kinase was examined. The mechanism of G protein-coupled receptor-mediated stimulation of this pathway is multifactorial and appears to be both receptor and cell-type dependent (26). For the ₂AR, coupling to G_i, internalization of the receptor and interaction with -arrestin is required for this receptor to activate the MAP kinase cascade (27). Less is known about ₂AR coupling to this pathway; however, it is clear that it is pertussis toxin-sensitive and that receptor internalization is not necessary (28). For the current studies, MAP kinase activation was assessed using quantitative immunoblots with an antibody specific for the activated (phosphorylated) form of extracellular signal-regulated kinase 1/2. The total amount of MAP kinase was not different between the two cell lines utilized (Fig. 5A). Agonist-promoted activation of MAP kinase was significantly different between the two receptors (Fig. 5), with results expressed both as the agonist-promoted fold increase over basal levels of activated MAP kinase and as the percent of the thrombin response. In five such experiments, MAP kinase activity in Del322-325-expressing cells in response to 10 µM epinephrine was 57.8 ± 7.0% of the WT_2CAR response (p < 0.005). When normalized to the thrombin response, epinephrine-stimulated MAP kinase activity was 37 ± 5.7% for the polymorphic receptor compared with 128 ± 10.0% for the wild-type _2CAR (p < 0.005).

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Stimulation of MAP kinase by wild-type and Del322-325 2CARs. Phosphorylation of MAP kinase was determined in CHO cells by quantitative immunoblotting with enhanced chemifluorescence using antibodies specific for phosphorylated extracellular signal-regulated kinase 1/2. The same blots were stripped and reprobed for total kinase expression, which was not significantly different between the two cell lines (A). Cells were studied after incubation with carrier (basal) (NT), 10 µM epinephrine (Epi), or 1 unit/ml thrombin (Thr). Results are depicted as the fold stimulation over basal normalized to the wild-type response (B) and the percent of the thrombin response (C). *, p < 0.005 compared with wild-type response (n = five experiments).

Recent studies have begun to elucidate specific functions for the _2CAR subtype. In situ mRNA and immunohistochemical analysis of _2CAR expression has revealed a distinct pattern of expression in rat brain and spinal cord (29, 30). _2CARs have been localized primarily in the neuronal perikarya and to a lesser extent in the proximal dendrites, with high levels of receptor expression detected in the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex (29). These data along with studies of genetically engineered mice indicate that the _2CAR subtype plays explicit roles in cognitive and behavioral functions. Studies of mice that overexpress or that have targeted inactivation of the _2CAR gene have shown that this receptor is involved in the regulation of spontaneous motor activity as well as agonist-induced regulation of body temperature and dopamine metabolism (2). In addition, results indicating that activation of _2CAR reduces hyperreactivity and impulsivity have also been reported (3). These studies show that lack of _2CAR expression is associated with increased startle reactivity, reduced prepulse inhibition of the startle reflex, and isolation-induced attack latency, whereas overexpression of _2CAR produces the opposite effects. Consistent with these data, in humans, the ₂AR agonist clonidine and the ₂AR antagonist idazoxan reduce and facilitate the acoustic startle response, respectively (31, 32). The role of _2CAR in modulating working memory has also been characterized (4). In these studies, _2CAR knockout mice performed less accurately in a delayed alternation task and displayed slowed motor initiation in the return phase of the task, supporting a role for the _2CAR in the cognitive aspect of response preparation. _2CAR knockout mice were also impaired in spatial and nonspatial water maze tests, thus supporting a role for this receptor in modulating cognitive functions (5), and alteration of _2CAR expression in transgenic mice has also been linked with behavioral despair development and changes in plasma corticosterone levels (11). Recent studies measuring [³H]norepinephrine release from central neurons and cardiac sympathetic nerves have shown that the frequency-release curves for _2CAR-deficient mice are rightward shifted compared with wild-type mice (9). Furthermore, the residual agonist-stimulated inhibition of [³H]norepinephrine release observed in _2AAR-deficient mice was not present in mice deficient in both _2A and _2CAR. Thus both subtypes are important in inhibiting neurotransmitter release at these sites. _2CAR mRNA or receptor protein has also been identified in other peripheral sites (10, 33, 34) with evidence in some cases indicative of postsynaptic functions (10).

The presence of functionally distinct polymorphic _2CARs may account for interindividual variability in physiological responses or may be the basis of differences in clinical characteristics of diseases where _2CAR function is important. In addition, the Del322-325 polymorphism could conceivably predispose individuals to the development of disease. The response to agonist or antagonist therapeutic agents may also vary depending on receptor genotype. In this regard individuals with Del322-325 might be more sensitive to antagonists because they have receptors that are less responsive to endogenous catecholamines. For agonists, the response or sensitivity would be predicted to be less for those with the polymorphic _2CAR due to its impaired coupling. Given the relatively high frequency of the polymorphism in healthy African-Americans (Table I), modification of a disease or drug response is more likely than predisposition to disease, although all these possibilities need to be explicitly tested. We and others have recently shown that functional polymorphisms of the ₂AR indeed appear to have one or more of the above effects in asthma, congestive heart failure, and obesity (35-37). Interestingly, Comings et al. (38) have found that increased levels of plasma norepinephrine levels in children with attention deficit hyperactivity disorder with learning disabilities were associated with polymorphisms near the coding regions of the _2A, _2C, and dopamine -hydroxylase genes.

In summary, we have identified a polymorphic _2CAR that consists of a deletion of four amino acids in the third intracellular loop of the receptor. Such a deletion has a significant impact on agonist-promoted formation of the active receptor-G protein ternary complex resulting in significantly altered functional signaling to inhibition of adenylyl cyclase, stimulation of inositol phosphate accumulation, and activation of MAP kinase. For all three effector pathways, the Del322-325 receptor displays markedly impaired coupling. The polymorphism is rare in Caucasians but is ~10-fold more prevalent in African-Americans with an allele frequency of 0.381. To our knowledge, this is the greatest racial difference in a polymorphism of any G protein-coupled receptor reported to date. Given the extreme phenotype, this locus should be considered a basis for interindividual variation in physiologic responses, disease predisposition or modification, and drug responsiveness.

	ACKNOWLEDGEMENTS

We thank Anil Menon for providing some of the DNA samples, Cheryl Theiss for cell culture, and Esther Getz for manuscript preparation.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants HL53436, ES06096, and HL41496.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: University of Cincinnati College of Medicine, 231 Bethesda Ave., Rm. 7507, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835; E-mail: stephen.liggett@uc.edu.

Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.M000796200

	ABBREVIATIONS

The abbreviations used are: ₂AR, ₂-adrenergic receptor; PCR, polymerase chain reaction; bp, base pair(s); _2CAR, ₂AR subtype C; Del322-325, _2CAR polymorphism resulting in deletion of amino acids 322-325; CHO, Chinese hamster ovary; MAP kinase, mitogen-activated protein kinase; WT, wild-type; GppNHp, 5'-guanylylimidodiphosphate.

	REFERENCES

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				R. Lazzara The congenital long QT syndrome: a mask for many faces. J. Am. Coll. Cardiol., March 4, 2008; 51(9): 930 - 932. [Full Text] [PDF]

				D. Kurnik, M. Muszkat, G. G. Sofowora, E. A. Friedman, W. D. Dupont, M. Scheinin, A. J.J. Wood, and C. M. Stein Ethnic and Genetic Determinants of Cardiovascular Response to the Selective {alpha}2-Adrenoceptor Agonist Dexmedetomidine Hypertension, February 1, 2008; 51(2): 406 - 411. [Abstract] [Full Text] [PDF]

				G. Kitsios and E. Zintzaras Genetic Variation associated with Ischemic Heart Failure: A HuGE Review and Meta-Analysis Am. J. Epidemiol., September 15, 2007; 166(6): 619 - 633. [Abstract] [Full Text] [PDF]

				N. Petrashevskaya and S. B. Liggett Tight control of adrenal medulla catecholamine release by {alpha}2C-adrenergic receptors influences susceptibility to heart failure Cardiovasc Res, September 1, 2007; 75(4): 631 - 633. [Abstract] [Full Text] [PDF]

				R. Gilsbach, M. Brede, N. Beetz, E. Moura, V. Muthig, C. Gerstner, F. Barreto, S. Neubauer, M. A. Vieira-Coelho, and L. Hein Heterozygous {alpha}2C-adrenoceptor-deficient mice develop heart failure after transverse aortic constriction Cardiovasc Res, September 1, 2007; 75(4): 728 - 737. [Abstract] [Full Text] [PDF]

				D. V. Menon, Z. Wang, P. J. Fadel, D. Arbique, D. Leonard, J.-L. Li, R. G. Victor, and W. Vongpatanasin Central Sympatholysis as a Novel Countermeasure for Cocaine-Induced Sympathetic Activation and Vasoconstriction in Humans J. Am. Coll. Cardiol., August 14, 2007; 50(7): 626 - 633. [Abstract] [Full Text] [PDF]

				BSCR Spring Meeting 2006. Heart, September 1, 2006; 92(9): e2 - e2. [Full Text] [PDF]

				J. G. Zaroff, L. Pawlikowska, J. C. Miss, S. Yarlagadda, C. Ha, A. Achrol, P.-Y. Kwok, C. E. McCulloch, M. T. Lawton, N. Ko, et al. Adrenoceptor Polymorphisms and the Risk of Cardiac Injury and Dysfunction After Subarachnoid Hemorrhage Stroke, July 1, 2006; 37(7): 1680 - 1685. [Abstract] [Full Text] [PDF]

				J.-L. Li, R. M. Canham, W. Vongpatanasin, D. Leonard, R. J. Auchus, and R. G. Victor Do Allelic Variants in {alpha}2A and {alpha}2C Adrenergic Receptors Predispose to Hypertension in Blacks? Hypertension, June 1, 2006; 47(6): 1140 - 1146. [Abstract] [Full Text] [PDF]

				K. M. Small, K. M. Brown, C. A. Seman, C. T. Theiss, and S. B. Liggett Complex haplotypes derived from noncoding polymorphisms of the intronless {alpha}2A-adrenergic gene diversify receptor expression PNAS, April 4, 2006; 103(14): 5472 - 5477. [Abstract] [Full Text] [PDF]

				V. Regitz-Zagrosek, B. Hocher, M. Bettmann, M. Brede, K. Hadamek, C. Gerstner, H. B. Lehmkuhl, R. Hetzer, and L. Hein {alpha}2C-Adrenoceptor polymorphism is associated with improved event-free survival in patients with dilated cardiomyopathy Eur. Heart J., February 2, 2006; 27(4): 454 - 459. [Abstract] [Full Text] [PDF]

				J. P. Etzel, B. K. Rana, G. Wen, R. J. Parmer, N. J. Schork, D. T. O'Connor, and P. A. Insel Genetic Variation at the Human {alpha}2B-Adrenergic Receptor Locus: Role in Blood Pressure Variation and Yohimbine Response Hypertension, June 1, 2005; 45(6): 1207 - 1213. [Abstract] [Full Text] [PDF]

				D. M. Kaye, B. Smirk, S. Finch, C. Williams, and M. D. Esler Interaction between cardiac sympathetic drive and heart rate in heart failure: Modulation by adrenergic receptor genotype J. Am. Coll. Cardiol., November 16, 2004; 44(10): 2008 - 2015. [Abstract] [Full Text] [PDF]

				N. W. Siecke and P. A. Insel Clarifying the effects of adrenergic receptor polymorphisms by measuring synaptic parameters J. Am. Coll. Cardiol., November 16, 2004; 44(10): 2016 - 2018. [Full Text] [PDF]

K. M. Small, J. Mialet-Perez, C. A. Seman, C. T. Theiss, K. M. Brown, and S. B. Liggett Polymorphisms of cardiac presynaptic {alpha}2C adrenergic receptors: Diverse intragenic variability with haplotype-specific functional effec