Polymorphic Deletion of Three Intracellular Acidic Residues of the alpha 2B-Adrenergic Receptor Decreases G Protein-coupled Receptor Kinase-mediated Phosphorylation and Desensitization*

Kersten M. Small, Kari M. Brown, Susan L. Forbes, and Stephen B. LiggettDagger

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

Received for publication, September 5, 2000, and in revised form, October 25, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

A polymorphic variant of the human alpha 2B-adrenergic receptor (alpha 2BAR), which consists of a deletion of three glutamic acids (residues 301-303) in the third intracellular loop was found to be common in Caucasians (31%) and to a lesser extent in African-Americans (12%). The consequences of this deletion were assessed by expressing wild-type and the Del301-303 receptors in Chinese hamster ovary and COS cells. Ligand binding was not affected, although a small decrease in coupling efficiency to the inhibition of adenylyl cyclase was observed with the mutant. The deletion occurs within a stretch of acidic residues that is thought to establish the milieu for agonist-promoted phosphorylation and desensitization of the receptor by G protein-coupled receptor kinases (GRKs). Agonist-promoted phosphorylation studies carried out in cells coexpressing the alpha 2BARs and GRK2 revealed that the Del301-303 receptor displayed ~56% of wild-type phosphorylation. Furthermore, the depressed phosphorylation imposed by the deletion was found to result in a complete loss of short term agonist-promoted receptor desensitization. Thus the major phenotype of the Del301-303 alpha 2BAR is one of impaired phosphorylation and desensitization by GRKs, and thus the polymorphisms renders the receptor incapable of modulation by this key mechanism of dynamic regulation.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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alpha 2-Adrenergic receptors (alpha 2AR)1 are cell surface receptors for catecholamines that bind to the Gi/Go family of G proteins, coupling to multiple effector systems including inhibition of adenylyl cyclase activity (1). alpha 2AR are widely expressed within the central and peripheral nervous system (2-4) and participate in a broad spectrum of physiologic functions such as regulation of blood pressure both centrally and within the vasculature, sedation, analgesia, regulation of insulin release, renal function, and cognitive and behavioral functions (5-12). Three human alpha 2AR subtypes have been cloned and characterized (alpha 2A, alpha 2B, and alpha 2C). The alpha 2BAR has a distinct pattern of expression within the brain, liver, lung, and kidney, and recent studies using gene knockouts in mice have shown that disruption of this receptor effects mouse viability (13), blood pressure responses to alpha 2AR agonists (13), and the hypertensive response to salt loading (14).

Like the alpha 2AAR subtype (15, 16), the alpha 2BAR undergoes short term agonist promoted desensitization (17). This desensitization is due to phosphorylation of the receptor, which evokes a partial uncoupling of the receptor from functional interaction with Gi/Go (18, 19). Such phosphorylation appears to be due to G protein-coupled receptor kinases (GRKs), a family of serine/threonine kinases that phosphorylate the agonist-occupied conformations of many G protein-coupled receptors (20). The process serves to finely regulate receptor function providing for rapid adaptation of the cell to its environment. Desensitization may also limit the therapeutic effectiveness of administered agonists. For the alpha 2BAR, phosphorylation of serines/threonines in the third intracellular loop of the receptor is dependent on the presence of a stretch of acidic residues in the loop that appears to establish the milieu for GRK function (18). In this report we delineate the phenotype of a common polymorphism of the alpha 2BAR (21, 22), which consists of a deletion of three glutamic acid residues in this region; such a variation has a pronounced effect on receptor phosphorylation leading to a loss of agonist-promoted desensitization.


    MATERIALS AND METHODS
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Polymorphism Detection-- The sequence encoding the third intracellular loop of the human alpha 2BAR (GenBankTM accession number AF005900) was examined for polymorphic variation by performing polymerase chain reactions (PCRs) to amplify this portion of the cDNA from genomic DNA derived from blood samples. In this paper the adenine of the initiator ATG codon of the open reading frame of the receptor 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 39 normal individuals was utilized. Two overlapping fragments encompassing the third intracellular loop region were generated using the following primer pairs: fragment 1 (534 bp), 5'-GCTCATCATCCCTTTCTCGCT (sense) and 5'-AAAGCCCCACCATGGTCGGGT (antisense) and fragment 2 (588 bp), 5'-CTGATCGCCAAACGAGCAAC (sense) and 5'-AAAAACGCCAATGACCACAG (antisense). The 5' end of each sense and antisense primer also contained sequences corresponding to the M13 forward (5'-TGTAAAACGACGGCCAGT) and M13 reverse (5'-CAGGAAACAGCTATGACC) universal sequencing primers, respectively. The PCR reactions consisted of ~100 ng of genomic DNA, 5 pmol of each primer, 0.8 mM dNTPs, 10% Me2SO, 2.5 units of Platinum Taq DNA polymerase (Life Technologies, Inc.), 20 µl of 5× buffer J (Invitrogen) in a 100-µl 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, 58 °C (fragment 1) or 60 °C (fragment 2) for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. PCR reactions were purified using the QIAquick PCR purification system (Qiagen), and automated sequencing of both strands of each PCR product was performed using Applied Biosystems 370 sequencer using dye primer methods. As discussed below, a 9 bp in-frame deletion beginning at nucleotide 901 was detected that resulted in a loss of three glutamic acid residues at amino acid positions 301-303. Thus, this polymorphism was denoted Del301-303. No other nonsynonymous or synonymous polymorphisms were identified. PCR amplification of 209- and 200-bp fragments encompassing this polymorphic region allowed screening of additional DNA samples whose genotypes were distinguished by size when run on 4% Nuseive agarose gels. PCR conditions were the same as described above except that buffer F was used with the following primers: 5'-AGAAGGAGGGTGTTTGTGGGG (sense) and 5'-ACCTATAGCACCCACGCCCCT (antisense).

Constructs and Cell Transfection-- To create the polymorphic alpha 2BAR construct, a 1585-bp PCR product encompassing the alpha 2BAR gene was amplified from a homozygous deletion individual using the following primers: 5'-GGCCGACGCTCTTGTCTAGCC (sense) and 5'-CAAGGGGTTCCTAAGATGAG. This fragment was digested and subcloned into the XcmI and BamHI sites of the wild-type alpha 2BAR sequence in the expression vector pBC12BI (17). The integrity of the construct was verified by sequencing. Chinese hamster ovary cells (CHO-K1) were stably 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 (23). Selection of positive clones was carried out in 1.0 mg/ml G418, and expression of the alpha 2BAR from individual clonal lines was determined by radioligand binding as described below. Several clonal lines with matched expression levels between 500 and 1000 fmol/mg were utilized as indicated. 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. For phosphorylation experiments, receptors were epitope tagged with the influenza hemagglutinin nonopeptide (YPYDVPDYA) at the amino terminus. This was accomplished by constructing vectors using the above constructs with insertions of in-frame sequence encoding the peptide using PCRs essentially as described previously (24). Tagged receptors were expressed at ~15 pmol/mg, along with GRK2 (beta ARK1), in COS-7 cells using a DEAE Dextran technique as described (16).

Adenylyl Cyclase Activities-- alpha 2AR inhibition of adenylyl cyclase was determined in membrane preparations from CHO cells stably expressing the two receptors using methods similar to those described previously (25). Briefly, cell 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 [alpha -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. Activities were measured in the presence of water (basal), 5 µM forskolin, and 5 µM forskolin with the indicated concentrations of agonists. 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. Results are expressed as percentages of inhibition of forskolin-stimulated activity. For desensitization experiments, cells were pretreated for 30 min at 37 °C with medium alone or with medium containing 10 µM norepinephrine, placed on ice, and washed five times with cold phosphate-buffered saline prior to membrane preparation. Desensitization of alpha 2BAR is manifested by a shift to the right in the dose-response curve for the inhibition of adenylyl cyclase (i.e. increase in EC50) without a significant change in the maximal response (17-19). To quantitate the magnitude of this desensitization, the inhibitory response under control conditions at a submaximal concentration of agonist (the EC50) in the assay was determined from the curve and compared with the response to this same concentration from membranes derived from cells exposed to norepinephrine. This method has been previously validated (26) in several G protein-coupled receptor systems.

Radioligand Binding-- Expression of mutant and wild-type alpha 2BAR was determined using saturation binding assays as described (25, 27) with [3H]yohimbine or [125I]aminoclonidine with 10 µM phentolamine or 10 µM yohimbine, respectively, 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 absence or presence of guanine nucleotide for 30 min at 25 °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 Whatman GF/C glass fiber filters.

Intact Cell Receptor Phosphorylation-- Transiently transfected COS-7 cells expressing equivalent levels of each receptor were grown to confluence and incubated with [32P]orthophosphate (~4 mCi/100-mm plate) for 2 h at 37 °C in 5% CO2 atmosphere. Cells were then incubated in the presence or absence of 100 µM norepinephrine for 15 min, washed five times with ice-cold phosphate-buffered saline, and solubilized in 1 ml of a buffer containing 1% Triton X-100, 0.05% SDS, 1 mM EDTA, and 1 mM EGTA in phosphate-buffered saline, by rotation in a microcentrifuge tube for 2 h at 4 °C. This and all subsequent steps included the protease inhibitors benzamidine (10 µg/ml), soybean trypsin inhibitor (10 µg/ml), aprotinin (10 mg/ml), and leupeptin (5 µg/ml) and the phosphatase inhibitors sodium fluoride (10 mM) and sodium pyrophosphate (10 mM). (A separate flask was scraped in 5 mM Tris/2 mM EDTA, and the membranes were prepared for radioligand binding as described above.) Unsolubilized material was removed by centrifugation at 40,000 × g at 4 °C for 10 min. The hemagglutinin epitope tagged alpha 2BARs were immunoprecipitated using an anti-hemagglutinin high affinity monoclonal antibody (Roche Molecular Biochemicals) as described previously (24). Briefly, solubilized material was preincubated for 2 h at 4 °C with protein G-Sepharose beads to remove nonspecific binding. The supernatant was then incubated with protein G-Sepharose beads and a 1:200 dilution of antibody for 18 h at 4 °C. Following immunoprecipitation, the beads were washed three times by centrifugation and resuspension and then incubated at 37 °C for 1 h in SDS sample buffer. Proteins in the supernatant were then fractionated on a 10% SDS-polyacrylamide gel with equal amounts of receptor (based on radioligand binding) loaded in each lane. Signals were visualized and quantitated using a Molecular Dynamics PhosphorImager with ImageQuant Software.

Miscellaneous-- Protein determinations were by the copper bicinchoninic acid method (28). 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 by Fisher's exact test. Comparisons of results from biochemical studies were by t tests, and significance was considered when p < 0.05. Data are provided as the means ± S.E.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
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Sequence analysis of the third intracellular loop of the alpha 2BAR gene from 78 chromosomes revealed a single sequence variant. This consisted of an in-frame 9-bp deletion (GAAGAGGAG) beginning at nucleotide 901 (Fig. 1A) that results in loss of three glutamic acid residues at amino acid positions 301-303 of the third intracellular loop of the receptor (Fig. 2). Using the rapid detection method (Fig. 1B), allele frequencies were determined in a larger population. The frequencies of the wild-type and the Del301-303 polymorphic alpha 2BAR are shown in Table I. The deletion polymorphism is more common in Caucasians than African-Americans, with allele frequencies of 0.31 and 0.12, respectively. The distribution of homozygous and heterozygous alleles in either population was not different than that predicted from the Hardy-Weinberg equilibrium (p > 0.8).



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Fig. 1.   Identification of the human alpha 2BAR variant. Shown in A and B are representative automated sequence chromatograms identifying a deletion of the nucleotides GAAGAGGAG. In C, a rapid screening technique identifies homozygous and heterozygous PCR products by size.



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Fig. 2.   Localization of the alpha 2BAR polymorphism. Shown are the fifth and sixth transmembrane spanning domains (TMD) and the third intracellular loop of the receptor.


                              
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Table I
Frequency of the alpha 2BAR Del301-303 polymorphism

The consequences of this polymorphism on ligand binding and receptor function were evaluated by stably expressing the human wild-type alpha 2BAR and the Del301-303 receptor in CHO cells (Table II). Saturation radioligand binding studies revealed a small but statistically significant lower affinity for the alpha 2AR antagonist [3H]yohimbine for Del301-303 compared with the wild-type receptor (Kd = 5.1 ± 0.2 versus 3.8 ± 0.3 nM, respectively, n = 5, p < 0.05). Agonist (epinephrine) competition binding experiments carried out in the presence of quanosine 5'(beta -alpha -imido)triphosphate revealed a small increase in the Ki for the polymorphic receptor (285 ± 8.7 versus 376 ± 66 nM, n = 5, p < 0.05). In similar studies carried out in the absence of guanine nucleotide, two-site fits were obtained for both receptors with no differences in the KL or the percentage of receptors in the high affinity state (RH; Table II). However, a trend toward an increased KH was observed with the Del301-303 mutant. These results prompted additional studies with the partial agonist radioligand [125I]aminoclonidine. Saturation binding studies (in the absence of quanosine 5'(beta -alpha -imido)triphosphate) with concentrations of the ligand from 0.2 to 4 nM revealed a single site with a Kd of ~1 nM as reported by others (27). Comparison of the wild-type alpha 2BAR and the Del301-303 receptor revealed essentially identical Kd values for [125I]aminoclonidine (1.33 ± 0.12 versus 1.22 ± 0.07 nM, respectively). Taken together, the data suggest that there is little, if any, effect of the deletion in the third intracellular loop on the conformation of the ligand binding pocket within the transmembrane spanning domains.


                              
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Table II
Ligand binding properties of wild-type and Del301-303 alpha 2BAR expressed in CHO cells
Saturation binding isotherms and competition studies were carried out with membranes from CHO cells expressing equivalent levels of receptor.

To address the functional consequences of the mutation, studies examining agonist-promoted inhibition of forskolin-stimulated adenylyl cyclase activities were carried out in lines expressing the wild-type alpha 2BAR and the Del301-303 receptor at densities of 626 ± 54 and 520 ± 82 fmol/mg (n = 7, p > 0.05). The results of these studies are shown in Table III. As can be seen, the Del301-303 receptor displayed less inhibition of adenylyl cyclase (23.4 ± 2.2%) compared with wild-type alpha 2BAR (28.5 ± 1.6%, p < 0.05). Furthermore, the polymorphic receptor had a greater EC50 (19.6 ± 5.5 versus 7.9 ± 2.1 µM, p < 0.01). Thus, the loss of the three glutamic acids in the third intracellular loop, which is known to contain regions important for G protein coupling, results in a modest decrease in agonist-mediated receptor function.


                              
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Table III
Adenylyl cyclase activities of the wild-type and Del301-303 alpha 2BAR expressed in CHO cells
Adenylyl cyclase activities were determined in membranes in response to forskolin (5 µM) and forskolin plus various concentrations of norepinephrine. See also Fig. 3.

The deletion polymorphism occurs in a highly acidic stretch of amino acids (EDEAEEEEEEEEEEEE) within the third intracellular loop of alpha 2BAR (Fig. 2). The structural importance of this region has been previously assessed and shown to be critical for short term agonist-promoted receptor phosphorylation leading to desensitization (18). These data and reports by others (29) suggest that this acidic environment is necessary for receptor phosphorylation by GRKs. Therefore, to investigate the consequences of this deletion polymorphism on receptor desensitization, agonist-promoted inhibition of adenylyl cyclase activity was determined in membranes from CHO cells expressing the wild-type and Del301-303 receptor after pretreatment with norepinephrine. In these experiments, cells were incubated with medium alone or medium containing agonist (10 µM norepinephrine) for 30 min at 37 °C and subsequently extensively washed at 4 °C; membranes were prepared, and agonist-mediated inhibition of forskolin stimulated adenylyl cyclase activity was determined. As described previously (17) and shown in Fig. 3 and Table II, desensitization of wild-type alpha 2BAR expressed in CHO cells is manifested by an increase in the EC50 for agonist-mediated inhibition of adenylyl cyclase. Analysis of composite curves derived from four independent experiments shows an increase from 7.4 to 29.4 µM for the wild-type alpha 2BAR. In contrast, there was no change in the EC50 for the deletion receptor following agonist pretreatment (29.5 µM versus 31.2 µM). Desensitization was quantitated by examining adenylyl cyclase activities at a submaximal concentration of agonist (the EC50 for the control condition). At this concentration, wild-type alpha 2BAR inhibited adenylyl cyclase activity by 16.5 ± 3.9%; with agonist pre-exposure, inhibition at this same concentration of agonist was 7.6 ± 2.3% (n = 4, p < 0.05, Fig. 3C), amounting to ~54% desensitization of receptor function. Submaximal inhibition of adenylyl cyclase for the Del301-303 receptor, however, was not different between control and agonist-treated cells (17.1 ± 3.0% versus 15.9 ± 1.7%, n = 4, p = ns). In another two cell lines with matched expression of ~600 fmol/mg, the same desensitization phenotypes for wild-type and the Del301-303 polymorphic receptor were observed (data not shown).



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Fig. 3.   The Del301-303 alpha 2BAR fails to undergo short term agonist-promoted desensitization. Cells in culture expressing the two receptors were exposed to vehicle or 10 µM norepinephrine for 30 min at 37 °C and washed extensively; membranes were prepared, and adenylyl cyclase activities were determined as described under "Experimental Procedures." In A and B, results of full dose-response studies are shown, which reveal that whereas the wild type undergoes desensitization manifested as a rightward shift in the curve, the Del301-303 mutant does not. In C, the percentage of inhibition of adenylyl cyclase at a submaximal concentration of norepinephrine in the assay (the EC50 of the control membranes) is shown for both conditions, indicating an ~54% desensitization of wild-type alpha 2BAR. The Del301-303 failed to display such desensitization. Results are from four independent experiments. See also Table III. *, p < 0.05 compared with control. WT, wild type.

We next performed whole cell phosphorylation studies of the wild-type and polymorphic alpha 2BAR under the same conditions used for desensitization. We hypothesized that agonist-promoted phosphorylation would be decreased in the polymorphic receptor. However, given that this receptor displays rightward-shifted dose-response curves for inhibition of adenylyl cyclase at base line, we also considered the possibility that the receptor is significantly phosphorylated in the basal state. Studies were carried out in cells cotransfected with the receptor and GRK2 (beta ARK1), a strategy that we have previously shown to be useful in identifying receptor-GRK interactions (30). The results of a representative study are shown in Fig. 4. The wild-type alpha 2BAR underwent a 5.84 ± 0.49-fold increase in phosphorylation with agonist exposure. In contrast, although the Del301-303 receptor displayed some degree of agonist-promoted phosphorylation, the extent was clearly less (3.28 ± 0.24-fold, p < 0.05 compared with wild type). Basal phosphorylation was equivalent between the two receptors.



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Fig. 4.   The Del301-303 alpha 2BAR has impaired agonist-promoted phosphorylation. Cells coexpressing each receptor and GRK2 were incubated with [32P]orthophosphate, exposed to 10 µM norepinephrine for 15 min, and receptor purified by immunoprecipitation as described under "Experimental Procedures." Shown is an autoradiogram from a single experiment representative of four performed (see text for mean results). NE, norepinephrine.

It is interesting to note that this partial loss of phosphorylation results in a receptor that fails to undergo any degree of functional desensitization. Although it might seem reasonable to assume that such phosphorylation would be associated with some degree of desensitization, several previous studies with the alpha 2AAR and alpha 2BAR subtypes indicate that full (i.e. wild type) phosphorylation is necessary for the desensitization process (16, 18, 24). For the alpha 2AAR, we have shown that four serines in the third intracellular loop are phosphorylated after agonist exposure (16). Removal of serines by alanine substitution mutagenesis results in a proportional decrease in phosphorylation. Such partial phosphorylation (compared with wild type), however, was found to be insufficient to cause any detectable desensitization. In a previous study of the alpha 2BAR, we deleted and substituted the entire aforementioned acidic region (18). Agonist-promoted phosphorylation was reduced by ~50% in this mutant, and desensitization was ablated. These results are entirely consistent with the current work, where a restricted deletion resulted in a decrease in phosphorylation and a complete loss of desensitization. Finally, we have also recently shown that a chimeric alpha 2A/alpha 2CAR, which undergoes agonist-promoted phosphorylation, fails to exhibit desensitization (24). Taken together with our current work, these results indicate that the conformation of the third loop evoked by GRK mediated phosphorylation which provides for the binding of arrestins (which is the ultimate step that imparts uncoupling) is highly specific. Thus, a precise phosphorylation-dependent conformation is apparently required for arrestin binding to alpha 2AR and subsequent functional desensitization. Perturbations of the milieu can thus have significant functional consequences, as occurs with the Del301-303 polymorphic alpha 2BAR.

Thus, the major signaling phenotype of the alpha 2BAR Del301-303 polymorphism is one of decreased agonist-promoted phosphorylation that results in a complete loss of the ability for the receptor to undergo agonist-promoted desensitization. In addition, the polymorphism imposes a small decrease in receptor coupling. The potential physiologic consequences of the polymorphism could be related to either or both of the above phenotypes. A receptor that fails to undergo desensitization would be manifested as static signaling despite continued activation of the receptor by endogenous or exogenous agonist. Such a lack of regulation by agonist may perturb the dynamic relationship between incoming signals and receptor responsiveness that maintains homeostasis under normal or pathologic conditions. Recently, Gavras and colleagues (14) have shown that alpha 2B -/+ mice fail to display a hypertensive response to salt loading after subtotal nephrectomy. Thus, a polymorphic alpha 2BAR that fails to desensitize (i.e. does not display regulatable function) may predispose to salt-sensitive hypertension. Regarding the therapeutic response to alpha 2AR agonists, the phenotype of the Del301-303 receptor indicates that individuals with this polymorphism would display little tachyphylaxis to continued administration of agonists. In addition, the initial response to agonist might also be reduced based on the somewhat depressed coupling of the Del301-303 receptor.

Until recently, it has been difficult to differentiate alpha 2BAR function from the other two subtypes in physiologic studies. With the development of knockout mice lacking each alpha 2AR subtype (5, 6, 13, 31), certain functions can now be definitively attributed to specific subtypes. Characterization of the alpha 2BAR knockout mouse has indicated that the alpha 2BAR subtype is expressed on vascular smooth muscle and is responsible for the hypertensive response to alpha 2AR agonists (13). This indicates that vascular alpha 2BAR contribute to overall vascular tone and thus participate in systemic blood pressure regulation. This role may be more important, however, during adaptive conditions, such as salt loading, because resting blood pressure is normal in the heterozygous alpha 2B -/+ mice (14). Whether the alpha 2B -/- mice have altered resting blood pressures has not been studied in detail because of high perinatal lethality of the homozygous knockout (13). However, neither the region of chromosome 2 near the alpha 2BAR coding sequence nor the deletion polymorphism has been linked or associated with hypertension (21, 22, 32). However, no studies have assessed whether the polymorphism is associated with salt-sensitive hypertension or other phenotypes, or the response to alpha 2AR agonist.

In summary, we have delineated the signaling phenotype of a polymorphism of the alpha 2BAR that results in a deletion of three glutamic acids in the third intracellular loop of the receptor. The polymorphism is prevalent in the human population, with a frequency that is ~2-fold greater in Caucasians as compared with African-Americans. The polymorphic receptor displays wild-type agonist binding affinity but a small decrease in function in the resting state. However, the major phenotype is a significant decrease in agonist-promoted phosphorylation by GRKs, which results in a receptor that fails to display agonist-promoted desensitization. To our knowledge this is the first polymorphism of any G protein-coupled receptor to affect GRK-mediated phosphorylation.


    ACKNOWLEDGEMENT

We thank Esther Getz for manuscript preparation.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL52318 and ES06096.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF316895.

Dagger To whom correspondence should be addressed: University of Cincinnati College of Medicine, 231 Albert Sabin Way, Rm. G062, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835; E-mail: stephen.liggett@uc.edu.

Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008118200


    ABBREVIATIONS

The abbreviations used are: AR, adrenergic receptor(s); GRK, G protein-coupled receptor kinase; PCR, polymerase chain reaction; bp, base pair(s); CHO, Chinese hamster ovary.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES


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