The presence of β2-adrenoceptors sensitizes α2A-adrenoceptors to desensitization after chronic epinephrine treatment

Background In addition to the regulation of blood pressure, α2- and β-adrenoceptor (AR) subtypes play an important role in the modulation of noradrenergic neurotransmission in the human CNS and PNS. Several studies suggest that the α2-AR responsiveness in cells and tissues after chronic epinephrine (EPI) or norepinephrine (NE) exposure may vary, depending on the β-AR activity present there. Recently, we reported that in BE(2)-C human neuroblastoma cells (endogenously expressing α2A- and β2-AR), chronic EPI treatment (300 nM) produced a dramatic β-adrenoceptor-dependent desensitization of the α2A-AR response. The aim of this study is to determine if stable addition of a β2-AR to a second neuroblastoma cell line (SH-SY5Y), that normally expresses only α2A-ARs that are not sensitive to 300 nM EPI exposure, would suddenly render α2A-ARs in that cell line sensitive to treatment with the same EPI concentration. Methods These studies employed RT-PCR, receptor binding and inhibition of cAMP accumulation to confirm α2-AR subtype expression. Stable clones of SH-SY5Y cells transfected to stably express functional β2-ARs (SHβ2AR4) were selected to compare sensitivity of α2-AR to EPI in the presence or absence of β2-ARs. Results A series of molecular, biochemical and pharmacological studies indicated that the difference between the cell lines could not be attributed to α2-AR heterogeneity. We now report that after transfection of functional β2-AR into SH-SY5Y cells (SHβ2AR4), chronic treatment with modest levels of EPI desensitizes the α2A-AR. This effect results from a β2-AR dependent down-regulation of native α2A-ARs by EPI accompanied by enhanced translocation of GRK2 and GRK3 to the membrane (required for GRK-mediated phosphorylation of agonist-occupied receptors). Conclusion This study further supports the hypothesis that the presence of the β-AR renders the α2A-AR more susceptible to desensitization with physiological levels of EPI.


Background
Studying changes in α 2 -adrenoceptor (AR) signaling is important for understanding the development and/or manifestation for several CNS (cerebral ischemia, pain, depression) and PNS disorders (hypertension and cardiac dysfunction). Under physiological conditions, norepinephrine and epinephrine (NE and EPI, respectively) activate the α 2 -AR along with other members of the AR family, which also includes α 1 -and β-ARs. The α 2 -and β-ARs are often co-expressed on the same cell surface. Upon activation by NE and EPI, the independent signals initiated by the α 2 -and β-ARs often converge to regulate specific physiological endpoints such as insulin release [1], maintenance of uterine smooth muscle tone [2], and noradrenergic transmission in the CNS and PNS [3,4]. The α 2 -and β-ARs regulate many of these physiological mechanisms by mediating opposing actions on adenylyl cyclase; α 2 -AR inhibits while β-AR stimulates the adenylyl cyclase pathway.
Continuous exposure to catecholamines leads to a declining receptor response, a phenomenon called desensitization. The process of desensitization generally includes receptor phosphorylation, internalization, and down-regulation. Unlike other members of the AR family, the α 2A -AR subtype does not readily down-regulate. Since this subtype is the dominant α 2 -AR in the CNS and mediates the "classical effects" of α 2 -ARs which include hypotension, sedation, and antinociception [5,6], numerous studies have focused on the regulatory mechanisms of the α 2A -AR. In cultured cell lines expressing either native α 2A -AR [7] or recombinantly over-expressed α 2A -AR [8,9], supraphysiological concentrations of EPI (100 μM) and NE (30 μM) were required to produce long-term α 2A -AR desensitization. The waning α 2A -AR signal is attributed primarily to down-regulation of the receptor and/or phosphorylation of the agonist occupied receptor by G-protein coupled receptor kinases (GRK), specifically GRK2 and GRK3 [10,11]. Previous studies suggest that either of these two α 2A -AR desensitization mechanisms require supra-physiological (μM) concentrations of agonist [10,[12][13][14].
However, our recent studies in the BE(2)-C human neuroblastoma cell line suggest that when β-ARs are present on the same cells lower, more physiologically relevant, concentrations of EPI (300 nM) are able to desensitize the α 2A -AR following chronic (24 hr) treatment [15]. In the absence of β-ARs, α 2A -AR desensitization occurs only with supra-physiological concentrations of EPI, if it occurs at all [15]. Concurrent activation of the β-AR and α 2A -AR also prompts down-regulation of cell surface α 2A -ARs while specifically up-regulating the expression of GRK3 within BE(2)-C cells [15]. Enhanced GRK3 expression plays a prominent role, as it is required for both β-ARdependent α 2A -AR desensitization and down-regulation [15,16]. Recently we reported similar findings for the α 2B -AR subtype in mouse neuroblastoma cells [17][18][19].
Since both α 2 -and β-ARs are often co-localized and share the same endogenous ligands, it is reasonable that the α 2A -AR response is regulated differently in the presence and absence of the β-AR. Indeed, evidence suggests that the α 2 -AR responsiveness in cells and tissues after chronic EPI or NE vary, depending on the β-AR activity present there [2,15,[20][21][22][23]. The aim of the present study is to compare α 2A -AR responsiveness after chronic EPI and NE treatment in non-β-AR expressing (wild-type SH-SY5Y, wt) human neuronal cells to α 2A -AR responsiveness in SH-SY5Y cells that have been stably transfected to express β 2 -AR (SHβ 2 AR4). In doing so, we hope to determine whether co-expression of the two ARs intrinsically produced this differential α 2A -AR regulation and whether enhanced expression of GRK3 is required for this regulation.

Characterization of the model system and establishment of the SHβ 2 AR4 cell line
Our first goal was to find a second model system that was similar to the BE(2)-C human neuroblastoma cell line (expressing modest levels of α 2A -AR), but that didn't express β-ARs. Kazmi and Mishra previously identified the SH-SY5Y cell line as expressing two α 2 -AR binding sites [24], while Parsley et al. [25] reported that it expressed a single AR subtype, α 2C , based upon functional and molecular studies. Since receptor expression varies depending on differentiation state and passage number, it was necessary to determine which α 2 -AR subtypes were expressed in our population of SH-SY5Y cells, using a combination of binding, functional, and molecular approaches. SH-SY5Y cells expressed α 2 -AR levels slightly greater than the level detectable in BE(2)-C cells (B max : SH-SY5Y, 67.6 ± 8.2 ; BE(2)-C, 40.8 ± 7.0 fmol/mg protein). According to nonlinear and linear regression analysis of saturation binding, the data best fit a single-site model in SH-SY5Y cells, as observed previously in BE(2)-C cells. Rauwolscine and yohimbine competed for specific [ 3 H]rauwolscine binding to SH-SY5Y cell membranes with higher affinity than prazosin, the α 2B/C -selective antagonist (Table 1; [24]). Apparent K i values of agonists and antagonists against [ 3 H]rauwolscine binding were determined for comparison with previously reported values in cells natively expressing α 2A -, α 2B , or α 2C -ARs (HT29 and BE(2)-C, NG108-15, OK; [15,26,27]) or cell lines expressing cloned α 2 C10, α 2 C2, and α 2 C4 [28]. Values obtained from binding studies in SH-SY5Y cells correlated only to values from BE(2)-C cells and showed the greatest similarity with those derived from native and cloned α 2A -AR-containing cell membranes (Table 2). These results are consistent with binding of [ 3 H]rauwolscine to an α 2A -AR in SH-SY5Y cells.
Functional studies were performed by measuring the ability of various α 2 -AR agonists to inhibit forskolin (10 μM)stimulated cAMP accumulation in intact cells. All α 2 -AR agonists inhibited forskolin-stimulated cAMP accumulation in a concentration-dependent manner; no stimulation of cAMP accumulation was noted in the absence of forskolin. Inhibition of cAMP accumulation by the α 2 -AR agonist UK14,304 (30 nM; Fig. 1) was completely reversed by 10 nM yohimbine, whereas the α 2B/C -selective antagonist ARC-239, at a concentration over 30-fold higher than that of the agonist, failed to reverse the actions of UK14,304. Thus, both binding and functional data support the classification of the α 2 -AR subtype in this neuroblastoma cell line as α 2A .
Since Parsley et al. [25] were unable to detect α 2A -AR RNA by performing RT-PCR with total RNA extract, we optimized our chances for detecting α 2A -AR RNA by generating RT-PCR products from SH-SY5Y mRNA using primer pairs selective for individual α 2 -AR subtypes (Table 3; [29,30]) or a primer pair that recognizes two α 2 -AR receptor subtypes distinguished by their restriction nuclease digestion products (Table 3; [30]). RT-PCR with α 2 C10/ C4 primers gave a 233 bp product specific for α 2A -and α 2C -ARs; restriction digestion of this fragment with BglII, that would specifically cleave α 2A -AR, resulted in two fragments of 117 bp and thereby established expression of α 2A -AR mRNA in SH-SY5Y cells. RT-PCR with α 2 C4 primers gave a 630 bp fragment, which was successfully digested by BstXI to produce three fragments of 271, 225, and 78 bp, consistent with the presence of an α 2C -AR gene product (Fig. 2). RT-PCR products were neither noted in samples lacking reverse transcriptase (-), nor were they produced with primers selective for α 2 C2 (α 2B -AR; data not shown). While SH-SY5Y cells express mRNA for both α 2A -and α 2C -ARs, it appears that the predominant functional α 2 -AR in our cell line is the α 2A -AR.
Since these cells appear to express α 2A -ARs with properties similar to those in BE(2)-C cells [15] but lack a β-AR, pcDNA 3.0 plasmid vector containing the human β 2 -AR gene was transfected into SH-SY5Y cells. Colonies of stable transfectants were selected and maintained by their resistance to G418 (600 μg/mL) and subsequently clonal populations of β 2 -AR-expressing SH-SY5Y cells (SHβ 2 AR) were screened for β-AR expression using [ 3 H]CGP-12177 for binding studies as described in Methods. Since BE(2)-C cells express very low levels of β 2 -AR (B max : 18.5 ± 6.2 fmol/mg protein), the SHβ 2 AR4 cell line that expressed 14.78 ± 4.19 fmol/mg protein of the β 2 -AR was selected for the subsequent studies. To ensure that the β-ARs were functional, the ability of isoproteranol (ISO) to stimulate  [40]. The Log(EC 50 ) values (concentration of the drug that produces 50% of the maximal inhibitory/stimulatory effect of that drug) were calculated by nonlinear regression analysis of the agonist concentration-response curves (n = 3-9) of each agonist. a Values from Kazmi and Mishra [24]. N.D., not determined; N.A., not applicable. Correlation coefficient values (r) were generated by comparing pK i values from Table 1 with previously published values for one-site models using Pearson correlation analysis (GraphPad Prism). The slope of the linear regression line is also included. Correlations were considered significant (*) if p ≤ 0.05.
cAMP accumulation was assessed ( Table 1). The α 2A -AR responses were also tested in this new cell line to confirm that α 2A -AR function had not been altered by the expression of the β 2 -AR (Table 1).

Chronic 300 nM EPI exposure induces α 2A -AR desensitization only in SH-SY5Y cells transfected with functional β-AR
To determine whether the presence of the β-AR influences α 2A -AR signaling, the ability UK14,304 to inhibit forskolin-stimulated cAMP accumulation was evaluated after wildtype (wt) and SHβ 2 AR4 cells were exposed to vehicle or the indicated concentration of agonist for 16-24 hr. Wt SH-SY5Y cells ( Fig 3A) require a 30-fold higher concentration of NE (30 μM) to desensitize the α 2A -AR signal than SHβ 2 AR4 cells (1 μM; Fig 3B). RT-PCR Products obtained from SH-SY5Y RNA using α 2 -AR subtype selective primers Figure 2 RT-PCR Products obtained from SH-SY5Y RNA using α 2 -AR subtype selective primers. RT-PCR experiments were performed as described in "Methods" using primer pairs recognizing α 2 C10/C4 (corresponding to α 2A and α 2C ) and α 2 C4 (corresponding to α 2C ) gene products ( Table 3). The reactions amplified fragments of the expected size from each set of primers. α 2 C10/C4 primers amplified 233 bp products from SH-SY5Y mRNA that were sensitive to digestion by BglII (specific for the α 2A product). Restriction digestion with BstXI of the 630 bp product of α 2C4 primer amplification gave three fragments of 271, 225 and 78 bp. All reactions were performed in the presence (+) or absence (-) of reverse transcriptase (RT) to rule out the possibility of DNA contamination. Lane M designates the 100 bp ladder; the 500 bp fragment is indicated by an arrow in each panel.
Reversal of the inhibitory effect of UK 14,304 on forskolin-stimulated cAMP accumulation responded to EPI (300 nM) and NE (1 μM) pretreatments as the parent SH-SY5Y cells did (Fig. 3C).
β 2 -AR signal is desensitized following exposure to 300 nM EPI To ensure that the β 2 -AR is functioning properly following catecholamine treatment, we evaluated the ability of ISO to stimulate cAMP accumulation over basal in SHβ 2 AR4 cells. The β 2 -AR signal is desensitized following chronic EPI but not NE treatment, consistent with the fact that NE has a low affinity for the β 2 -AR. Inclusion of propranolol (30 nM) inhibited EPI-induced β 2 -AR desensitization (p < 0.05; Fig. 4), but had no effect in the absence of EPI.

SHβ 2 AR4, but not wt SH-SY5Y cells
Our study in BE(2)-C cells suggests that β 2 -AR-induced α 2A -AR desensitization following long-term EPI exposure is due in part to down-regulation of the α 2 -ARs. To determine if the same mechanism is responsible for the EPIinduced α 2A -AR desensitization in SHβ 2 AR4 cells, changes in α 2A -AR expression following catecholamine treatment were evaluated. Specific binding was measured with a single concentration of radioligand. We, and others, have shown that this is sufficient for accurate assessment of changes in receptor number for the α 2A -AR [9,15]. Chronic exposure of SHβ 2 AR4 cells to 300 nM EPI downregulates the α 2A -ARs by 20% (p < 0.05; Fig. 5). The α 2A -AR down-regulation in this cell line, as in BE (2) Chronic 300 nM EPI down-regulates α 2A -AR in β 2 -AR-trans-fected, but not native, SH-SY5Y cells Figure 5 Chronic 300

Chronic EPI exposure does not alter GRK2 or GRK3 levels in whole cells but instead enhances GRK2 and GRK3 expression at the membrane in SHβ 2 AR4 cells
We previously established that EPI-induced α 2A -AR desensitization and down-regulation in BE(2)-C cells is mediated via β 2 -AR-dependent GRK3 up-regulation [15]. Therefore, GRK3 levels in whole cell SHβ 2 AR4 lysates were evaluated following 24 hr catecholamine treatments. Chronic EPI exposure altered neither GRK3 nor GRK2 levels in the transfected SH-SY5Y cell line (Table 4). Therefore, unlike results in BE(2)-C cells, increases in whole cell GRK3 levels do not contribute to the modest α 2A -AR desensitization or down-regulation observed in the SHβ 2 AR4 cells.
Although GRK3 levels in whole cell lysates remain unaltered in SHβ 2 AR4 cells, it is not known whether GRK3 recruitment to the membrane is regulated via chronic EPI treatment in that cell line. Since GRK2 and GRK3 have been shown to regulate α 2A -AR signaling [10], we wanted to determine whether the membrane recruitment of either GRK isoform was changed following chronic EPI exposure in SHβ 2 AR4 cells. GRK2 and GRK3 are cytosolic proteins that anchor to the membrane via interaction with free Gβγ subunits; thus both kinases translocate from the cytosol to the membrane to regulate receptor signaling upon activation. Taking this characteristic of GRK2 and GRK3 into account, the levels of both kinases in membrane fractions following chronic EPI exposure were evaluated. SHβ 2 AR4 cells exhibit an increase in membrane-associated GRK2 and GRK3 with 24 hr EPI treatment compared to vehicle (P < 0.05; Fig. 6). In SHβ 2 AR4 cells, the same propranolol concentration (30 nM) that inhibited EPI-induced α 2A -AR desensitization and down-regulation also attenuated EPIinduced increase in GRK2 and GRK3 content in the membrane fraction (P < 0.05; Fig. 6). In contrast, no increased translocation of GRKs by EPI treatment was observed in wt SH-SY5Y cells that do not express β 2 -ARs. Therefore, this increased GRK2 and GRK3 translocation to the membrane following prolonged EPI treatment in SHβ 2 AR4 cells is β 2 -AR dependent.

Discussion
The major finding of the present study is the confirmation (using a different approach) that sensitivity of α 2A -AR to desensitization following exposure to relatively low levels of epinephrine is significantly increased in cells expressing both α 2A -and β 2 -AR. The first evidence for this was recently reported in a human neuronal cell line endog-Chronic 300 nM EPI enhances expression of GRK3 and GRK2 at the membrane of SHβ 2 AR4 cells via β 2 -AR-depend-ent mechanism Figure 6 Chronic 300 nM EPI enhances expression of GRK3 and GRK2 at the membrane of SHβ 2 AR4 cells via β 2 -AR-dependent mechanism. Wildtype SH-SY5Y (Wt SH) and SHβ 2 AR4 cells were subjected to catecholamine treatment in the presence or absence of 30 nM propranolol. Isolation of the membrane fraction and immunoblotting for GRK2 and GRK3 was conducted as described in Methods. EPI exposure significantly increased the level of GRK3 and GRK2 expressed in the membrane fractions from SHβ 2 AR4 cells compared to vehicle-treated controls (*P < 0.05; n = 3). Inclusion of propranolol (P) with EPI treatment prevented the increased translocation of both GRK isoforms ( # P < 0.01 as compared to EPI treatment), while propranolol treatment alone was without effect. In contrast, EPI failed to increase mobilization of GRK to the plasma membrane of wt SH cells (n = 4-7). Data represent mean ± S.E.; comparisons were made by ANOVA with Tukey's post-hoc test. enously expressing α 2A -and β 2 -ARs. Alpha 2A -and β-ARs in BE(2)-C cells desensitized after chronic EPI (300 nM), but not NE (1 μM), treatment [15]. Interestingly, the α 2A -AR responsiveness in SH-SY5Y cells (an alternative human neuroblastoma cell line that does not express β-ARs) is not desensitized after chronic treatment with 300 nM EPI or 1 μM NE (Fig. 3).
Obviously, the difference in α 2 -AR sensitivity to lower concentrations of EPI could be due to several factors, including differences in the α 2 -AR subtypes expressed in each cell line. Since it is difficult to demonstrate with great certainty what α 2 -AR subtypes are present in a given cell or tissue by biochemical or pharmacological means only, we took a molecular approach to ascertain which subtypes might potentially be expressed based on the presence of mRNA encoding each subtype. SH-SY5Y cells contained mRNA for α 2A -and α 2C -ARs (Fig. 2). As we noted and as reported by others [25], no evidence for α 2B mRNA was found. This was further confirmed by Northern blot analysis (data not shown). Initially, total RNA isolated from SH-SY5Y cells did not produce the α 2A -AR PCR products using the antisense primer selective for α 2A -AR previously described [30]. Instead α 2A -AR RT-PCR product was obtained only with poly(A) mRNA. However, employing poly(A)-enriched mRNA in the RT-PCR did not yield an α 2B -AR RT-PCR product. Parsley et al. [25]) identified only α 2C -AR mRNA using total RNA isolated from SH-SY5Y cells; this observation may reflect the limitation associated with the α 2A -AR primers used for RT-PCR of total RNA, similar to what we encountered.
The rank order binding affinity of the various agonists and antagonists tested is in agreement with that previously reported in cells expressing recombinant [3,28,31] or native α 2A -ARs [26,32]. When we compared apparent pK i values for various α 2 -AR agonists and antagonists against binding to [ 3 H]rauwolscine in SH-SY5Y membrane homogenates with previously reported values, we saw a correlation only with those cells that expressed α 2A -ARs (Table 2). Another means of distinguishing between various α 2 -AR subtypes involves comparing the prazosin/ oxymetazoline (OXY) or OXY/yohimbine affinity ratios (Table 1; [4]). Prazosin/OXY (74.4) and OXY/yohimbine (1.95) ratios were within the range reported for native and recombinant α 2A -ARs, and differ by at least 10-fold from values reported for α 2C -AR (from [4]). The agonist potency series in SH-SY5Y cells also most closely parallels that reported for the α 2A [26][27][28]32]. The inhibitory effect of α 2 -AR agonists on cAMP production in SH-SY5Y cells is readily reversed in a concentration-dependent fashion by the antagonist yohimbine (Fig. 1); the failure of the selective α 2B/C antagonist ARC-239 to antagonize UK 14,304 is consistent with activation of α 2A -ARs in SH-SY5Y cells.
Therefore, our results strongly support the designation of the functional α 2 -AR in SH-SY5Y cells as α 2A .
The present study supports our previous findings that pretreatment with a modest EPI concentration readily desensitizes the α 2A -AR signal in the presence, but not in the absence, of the β 2 -AR. This conclusion is based on several results. First, in wt SH-SY5Y cells (no β 2 -AR), the α 2A -AR signal is not desensitized following 24 hr treatment with modest concentrations of EPI or NE (300 nM and 1 μM, respectively). Instead wt cells required chronic exposure to supra-physiological concentrations of catecholamines (30 μM NE and 100 μM EPI; data not shown) for desensitization of the α 2A -AR signal; supporting the fact that α 2A -ARs do not desensitize and/or down-regulate readily in response to low to moderate levels of EPI. Second, 300 nM EPI induces α 2A -AR desensitization only in SHβ 2 AR4 cells which express functional β 2 -AR. Finally, EPI-generated waning of the α 2A -AR response is not observed in transfected cells expressing the pcDNA plasmid vector minus the β 2 -AR gene. This observation suggests that introduction of the β 2 -AR, and not the vector, is responsible for the difference in the α 2A -AR signal between wt and SHβ 2 AR4 cells exposed chronically to modest EPI concentrations.
As previously observed in BE(2)-C cells [15], desensitization of α 2A -AR signal with 24 hr EPI exposure is due, in part, to down-regulation of the receptor in SHβ 2 AR4 cells. Chronic co-activation of both α 2A -and β 2 -AR is required for desensitization and down-regulation of the α 2A -AR in SHβ 2 AR4 cells as indicated by the following results. First, 300 nM EPI, but not 1 μM NE, produces α 2A -AR desensitization and down-regulation in the recombinant cell line. Lands et al. [33] established that EPI has equal affinity for α 2A -and β 2 -AR while NE has a higher affinity for the α 2 -AR than β 2 -AR; therefore, EPI activates both α 2A -and β 2 -ARs simultaneously while NE activates the α 2A -AR alone. It is evident that the modest EPI concentration readily activates the β 2 -AR since chronic pretreatment with 300 nM EPI, but not 1 μM NE, desensitized the β 2 -AR response. Second, the inclusion of the β 2 -AR blocker propranolol prevented EPI-induced α 2A -AR desensitization and down-regulation in β 2 -AR-transfected SH-SY5Y cells. This propranolol concentration (30 nM) is sufficient to prevent EPI activation of β 2 -AR as indicated by the inhibition of EPI-induced β 2 -AR desensitization.
Although chronic EPI treatment desensitized and downregulated α 2A -AR in both BE(2)-C and SHβ 2 AR4 cells, several differences were observed. First, a more profound loss of efficacy is observed following 24 hr EPI exposure in BE(2)-C cells as compared to SHβ 2 AR4 cells. The maximal inhibition of forskolin-stimulated cAMP accumulation by UK14,340 was reduced 54% in BE(2)-C, but only 27% in SHβ 2 AR4, cells following EPI treatment (Fig. 3). The greater down-regulation of α 2A -ARs observed in BE(2)-C versus SHβ 2 AR4 cells most likely accounts for the greater change in efficacy: in SHβ 2 AR4, chronic EPI treatment produces a 20% loss of α 2A -ARs while in BE(2)-C cells, there is a 60% α 2A -AR down-regulation (Fig. 5). This more profound α 2A -AR desensitization and down-regulation observed in BE(2)-C is mediated via the up-regulation of GRK3 [15]. The lack of GRK3 up-regulation in the SHβ 2 AR4 cells is the second major difference between the two cell lines. At present, it is unknown what prompts GRK3 up-regulation in BE(2)-C cells but not in the SHβ 2 AR4 cells. However, we have observed that ERK1/2 activation is required for this induction of GRK3 following chronic exposure of BE(2)-C cells to EPI [16]. Moreover, while α 2A -ARs do not readily activate this pathway in neuronal cells, and β-AR activation by ISO can activate ERK1/2 at high concentrations, we have observed that ERK1/2 activation by EPI at concentrations that up-regulate GRK3 appears to require the simultaneous activation of both α 2A -and β 2 -ARs. Conversely, the inability of transfected β-ARs to prompt ERK1/2 activation in SH-SY5Y cells could explain the lack of GRK3 up-regulation in SHβ 2 AR4 cells.
Even though total GRK3 levels are unaltered, GRKs play a role in β 2 -AR-regulated α 2A -AR signaling in SHβ 2 AR4 cells as indicated by several results. First chronic EPI treatment enhances localization of GRK2 and GRK3 to the membrane. As indicated previously, translocation of these two cytosolic kinases to the membrane is required for phosphorylation and subsequent desensitization of its receptor substrate, which in this study is the α 2A -AR. Second, addition of propranolol attenuated EPI-mediated translocation of both GRK isoforms. This same propranolol concentration also inhibited α 2A -AR desensitization and down-regulation as discussed above. Therefore, β 2 -AR coactivation with α 2A -AR is required for enhanced GRK2 and GRK3 translocation to the membrane and subsequent α 2A -AR desensitization and down-regulation.
Translocation of both GRK2 and GRK3 to the membrane following chronic EPI treatment in SHβ 2 AR4 cells differs from the selective translocation of GRK3 (but not GRK2) observed in BE(2)-C cells following the same treatment (unpublished observations). The selective GRK3 up-regulation in BE(2)-C cells could account for the enhanced GRK3 levels at the membrane in these cells since in a previous study increase in total GRK2 levels promoted increased GRK2 expression at the membrane [34]. It is unknown at present why chronic EPI treatment translocates both GRK2 and GRK3 in SHβ 2 AR4 cells, and not in BE(2)-C cells. A possible explanation for the difference in the GRK isoform translocation between the two cell lines is differences in the β subunit expressed and/or released upon EPI exposure. GRK2 and GRK3 require the βγ subunit of the G proteins to anchor to the membrane but GRK2 and GRK3 exhibit distinct binding preferences for individual β subunits [35]. The β 3 isoform preferentially binds GRK3 but not GRK2, whereas β 1 and β 2 bind equally to both GRK3 and GRK2 [35,36].

Conclusion
Based on results obtained in this series of experiments, we conclude that exposure to modest EPI concentrations readily desensitizes and down-regulates α 2A -ARs in the presence, but not in the absence, of a functional β-AR. The β-AR-dependent down-regulation of α 2A -ARs is modulated via GRKs. In BE(2)-C cells, chronic co-activation of βand α 2A -AR prompts enhanced expression of GRK3, but not GRK2, in whole cells [15] and membrane fractions. In contrast, EPI pretreatment of SH-SY5Y cells transfected with functional β 2 -ARs does not increase either GRK3 or GRK2 expression per se, but does increase translocation of GRK2 and GRK3 to the plasma membrane. Like α 2A -AR desensitization and down-regulation, this translocation of GRK2 and GRK3 in SHβ 2 AR4 cells is β-AR-dependent and thus presents an alternate mechanism for the regulation of the α 2A -ARs by β-ARs.
Cell culture SH-SY5Y (passages 37-55) human neuroblastoma cells (Dr. Robert A. Ross, Fordham University, Bronx, NY) were maintained in a humidified atmosphere (6% CO 2 :94% air) in a 1:1 mixture of Eagle's minimum essential medium with non-essential amino acids and Ham's F-12 that contains 10% fetal bovine serum, 100 U/ml penicillin G and 0.1 mg/ml streptomycin sulfate. Plates of cells greater than 60% confluence were used throughout the study.

Transfection
Plasmid cDNA with the human β 2 -AR gene (provided by Dr. Brian Knoll; University of Houston, Houston, TX) or vector alone was stably transfected into SH-SY5Y cells with the fuGENE 6 Transfecting Reagent (Roche). Ten positive clones were isolated by their resistance to 800 μg/ mL of G418 and maintained in media containing 600 μg/ mL of G418. SHβ 2 AR4 was selected for use in all experiments because it expressed similar levels of β 2 -ARs as that expressed natively in BE(2)-C cells; SHβ 2 AR4 expressed 14.78 ± 4.19 fmol/mg protein while BE(2)-C express 18.5 ± 6.2 fmol/mg protein [15]. This β 2 -AR level remained consistent to passage 12 in SHβ 2 AR4 cells. After passage 12, SHβ 2 ARs neither expressed β 2 -ARs nor maintained resistance to G418, suggesting that the cells no longer expressed the transfected plasmid.
ODNs [29,30] corresponded to sequences for the various human 1% Triton X-100, 50 mM KCl] in sterile distilled water. Reaction mixtures were overlayed with mineral oil and subjected to a hot start for 5 min at 95°C. DNA polymerase (2.5 U taq, 5 U/ μl, Promega, Madison, WI) was added to each reaction tube after the hot start, and the tubes were subjected to a PCR reaction of 30 cycles in a thermal cycler (MJ Research Inc., Watertown, MA) for 1 min at 94°C, 1.5 min at 55°C, and 2 min at 72°C with a final elongation step at 72°C for 7 min. Reaction products were separated by electrophoresis through 2% agarose gels and visualized by ethidium bromide staining. PCR products were isolated from the gel using a DNA extraction kit (Amicon Inc., Bedford, MA). Identity of the purified PCR products was confirmed by their susceptibility to digestion with restriction enzymes specific for each reaction product (see Table 1; [30]).

cAMP accumulation
To determine the effects of α 2 -AR agonists on forskolininduced cAMP accumulation, intact cells were incubated for 5 minutes at 37°C in HBSS buffer (in mM): NaCl (137), KCl (5), Na 2 HPO 4 (0.6), KH 2 PO 4 (0.4), NaHCO 3 (4), D-glucose (6), MgCl 2 (0.5), MgSO 4 (0.4) and CaCl 2 (1), containing the phosphodiesterase inhibitor IBMX (0.5 mM). In some experiments, antagonists also were included in this step. To prohibit oxidation, sodium ascorbate (0.11 mM) was included when assaying catecholamines. Upon addition of forskolin (10 μM) and agonist, assay tubes were incubated for an additional 10 min at 37°C. Removing the tubes to a boiling water bath for 5 min terminated the assay. All assays were performed in duplicate in a total volume of 0.5 ml. After boiling, samples were centrifuged for 5 min at 14000 × g, and cAMP levels from the supernatant fractions were determined in a [ 3 H]cAMP (0.8 pmol) binding assay as previously described [38]. β-AR-mediated stimulation of cAMP accumulation was performed in the same manner except that forskolin was not included in the assay mixture. Forskolin (10 μM) stimulated cAMP accumulation to 587 ± 88 pmol/mg protein (n = 46), 15-fold over basal levels (40.5 ± 2 pmol/mg protein).

Receptor binding Preparation of cell membranes
Cells were homogenized in 20 volumes of Tris-HCl buffer (50 mM, pH 7.4) containing NaCl (100 mM), Na 2 EDTA (1 mM) and PMSF (0.1 mM), and the membranes sedimented by centrifugation for 30 minutes at 34000 × g at 4°C. Pellets were resuspended in 0.32 M sucrose, and aliquots of the membrane fractions were stored frozen (-80°C) until use.

Saturation experiments
The level of α 2 -ARs in SH-SY5Y cell membranes (0.5 mg/ ml) was determined with various concentrations of [ 3 H]rauwolscine (60-80 Ci/mmol, 0.3 -12 nM) in a total volume of 1-2 ml in potassium phosphate buffer (50 mM, pH 7.4) containing MgSO 4 (5 mM) at 37°C for 45 min. Thereafter, 2 ml Tris-HCl (5 mM, pH 7.4, 4°C) was added to the homogenate to terminate the binding reaction and the contents of the tubes was filtered over #32 glass fiber filter strips (Schleicher & Schuell, Keene, NH) using a PHD cell harvester (Cambridge Technology, Cambridge, MA). The reaction tubes and the filter strips were rinsed twice with a further 2-3 ml of buffer. Levels of radioactivity were determined by scintillation spectroscopy in