The Second Intracellular Loop of the alpha 2-Adrenergic Receptors Determines Subtype-specific Coupling to cAMP Production*

(Received for publication, November 18, 1996)

Johnny Näsman Dagger §, Christian C. Jansson and Karl E. O. Åkerman Dagger

From the Dagger  Department of Physiology and Medical Biophysics, Uppsala University, BMC, Box 572, S-75123 Uppsala, Sweden and the  Department of Pharmacology and Clinical Pharmacology, University of Turku, FIN-20520 Turku, Finland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The alpha 2-adrenergic receptors (alpha 2-ARs), which primarily couple to inhibition of cAMP production, have been reported to have a stimulating effect on adenylyl cyclase activity in certain cases. When expressed in Spodoptera frugiperda Sf9 cells the alpha 2A subtype showed only inhibition of forskolin-stimulated cAMP production when activated by norepinephrine (NE), whereas the alpha 2B subtype displayed a biphasic dose-response curve with inhibition at low concentrations of NE and a potentiation at higher concentrations. To further investigate the subtype-specific coupling, we expressed a set of chimeric alpha 2A-/alpha 2B-ARs at similar expression levels in Sf9 cells to determine the structural domain responsible for the difference between the two subtypes. When the third intracellular loops were interchanged between alpha 2A and alpha 2B subtypes, the coupling specificity remained unchanged, indicating that this loop does not confer selectivity toward a stimulating response. A biphasic dose-response curve, typical for the alpha 2B subtype, could be seen when the second intracellular loop of the alpha 2B subtype was inserted into the alpha 2A subtype, suggesting that this loop is important for determining the subtype-specific coupling of alpha 2-ARs to cAMP production. Site-directed mutagenesis of non-conserved amino acids in the second intracellular loop of the alpha 2A subtype indicated that several residues are involved in the coupling specificity.


INTRODUCTION

The alpha 2-adrenergic receptors (alpha 2-ARs)1 are members of a large family of heptahelical receptors mediating the extracellular stimuli to the interior of the cell through G proteins. Three different subtypes of alpha 2-ARs, alpha 2A, alpha 2B, and alpha 2C, can be distinguished based on the affinity for selective ligands (1), and this subdivision has been confirmed with the molecular cloning of three separate genes (2-4). Funtional expression of cloned cDNAs in different cell types has shown that the alpha 2-ARs can mediate multiple cellular responses including inhibition or stimulation of adenylyl cyclase activity (5-9), activation of phospholipase A2 and D (5, 10), stimulation of phosphatidylinositol turnover (6), and mobilization of intracellular Ca2+ (11, 12).

In some cells, activation of alpha 2-ARs leads to a biphasic regulation of adenylyl cyclase activity with an inhibitory phase at low concentration of agonist and a stimulatory phase at higher concentrations (5, 7, 13, 15), whereas in other cells, the response is exclusively inhibitory (6, 8) or stimulatory (9, 13-15). The stimulation of cAMP production with alpha 2-AR agonists has also been shown in physiological systems with endogenous receptors (16, 17), suggesting that it is not a mere side effect seen in recombinant systems.

The seemingly controversial effect of alpha 2-AR activation on stimulation of adenylyl cyclase activity has been attributed to relatively high expression levels of the receptors (7), cell-type-specific expression of different types of adenylyl cyclases (14, 15), or overexpression of Gs protein (18). A direct interaction of alpha 2-ARs with Gs protein has been shown in Chinese hamster ovary cells (7), while in other cell types, the mechanism for stimulation seems less clear (9, 15, 19). In many cases, the stimulatory response has shown subtype selectivity, the stimulation being more pronounced with the alpha 2B subtype compared with the alpha 2A and alpha 2C subtypes (9, 13, 15). This suggests that the G protein coupling domains differ between the subtypes. Different cytoplasmic domains have been implicated in G protein activation and selection both in adrenergic receptors (20, 21) and other heptahelical receptors (22, 23). The aim of the present study was to investigate the apparent subtype-specific coupling of the mouse alpha 2-AR subtypes, alpha 2A and alpha 2B, when expressed in Spodoptera frugiperda (Sf9) cells at comparable receptor levels, and to delineate the receptor domain leading to potentiation of cAMP production.


EXPERIMENTAL PROCEDURES

Materials

[3H]Adenine (21 Ci/mmol) and [3H]RX821002 (48 Ci/mmol) were from Amersham Corp. (Buckinghamshire, UK). [14C]cAMP (309 mCi/mmol) was from DuPont NEN. Cholera toxin, isobutylmethylxanthine (IBMX), (-)-norepinephrine, pertussis toxin, propranolol, and quinacrine were from Sigma. Phentolamine and UK14,304 were from Research Biochemicals International (Natick, MA).

Cell Culture

Sf9 cells were maintained as suspension culture at 25-27 °C in TNM-FH medium (pH 6.3) supplemented with 10% fetal calf serum (Life Technologies, Inc., Paisley, UK), 100 units/ml penicillin (Nordvacc Media, Skärholmen, Sweden), and 100 µg/ml streptomycin (Nordvacc Media), and 2.5% amphotericin B (Life Technologies, Inc.). For expression, Sf9 cells were subcultured in monolayer and infected with recombinant baculoviruses at a multiplicity of infection of 2-5 for the indicated times.

Recombinant Baculoviruses

The cDNAs for the mouse alpha 2A- and alpha 2B-ARs and three mouse chimeric receptors, MCR1, -2, and -3, were a gift from Dr. B. Kobilka (Howard Hughes Medical Institute, Stanford University). All clones contained a hemagglutinin tag fused to the amino terminus of the receptor constructs. For generation of recombinant baculoviruses, the genes were subcloned into the baculovirus transfer vector plucGRBac1 (9) under the transcriptional regulation of the polyhedrin gene promoter. The transfer vectors were then used for cotransfection with wild-type Autographa Californica nuclear polyhedrosis virus DNA, and recombinant viruses were purified essentially as described by Jansson et al. (9).

To generate chimeric receptors MCR4 and MCR5, the cDNA clones for alpha 2A- and alpha 2B- ARs were transferred into the SmaI site of pBluescript (Stratagene, La Jolla, CA) in a KS orientation. A SapI restriction site was introduced into the alpha 2A sequence at nucleotide position 351 of the coding sequence (equivalent to the position of a SapI site in the alpha 2B cDNA) using standard PCR techniques (24). The sequence from the beginning of the alpha 2A gene to the novel SapI site was then inserted into pBluescript-alpha 2B cut with the same enzyme to generate pBluescript-MCR4.

MCR5 was constructed by isolating a DraIII-fragment (DraIII cuts the cDNAs of alpha 2A and alpha 2B at an equivalent position and the plasmid DNA at one position) from pBluescript-alpha 2A and ligating it into an isolated DraIII-fragment of pBluescript-MCR4 to generate pBluescript-MCR5. The sequences encoding MCR4 and MCR5 were cut out from the pBluescript constructs with EcoRI and XbaI and ligated into pFastBac1 digested with the same enzymes. For production of recombinant baculovirus, a BAC-TO-BAC baculovirus expression system kit (Life Technologies, Inc.) was used.The correctness of all plasmid constructs were checked by restriction enzyme mapping and partial dideoxy sequencing (25).

Site-directed mutagenesis of the alpha 2A sequence was performed using PCR as described (24). Mutated alpha 2A sequences were subcloned into pBluescript, and the mutations were verified by sequencing. The mutated sequences were subsequently transferred to pFastBac1 with EcoRI and NotI. Recombinant baculoviruses were generated using the BAC-TO-BAC baculovirus expression system kit.

Measurement of Cellular cAMP

Sf9 cells were plated on tissue culture dishes and allowed to attach for 1 h before infection with respective recombinant baculovirus for the indicated times. The cells were incubated with 5 µCi/ml [3H]adenine for 2-3 h and thereafter were scraped off, pelleted, and washed in MES-buffered medium (130 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl2, 4.2 mM NaHCO3, 7.3 mM NaH2PO4, 20 mM MES, 63 mM sucrose, 10 mM glucose, and 1 mM CaCl2, pH 6.3). The pellet was resuspended in the same buffer and devided into aliquots of about 106 cells/0.8 ml. The cells were preincubated with 0.5 mM IBMX, 100 µM propranolol, and 150 µM quinacrine for 10 min at 26 °C, and then forskolin (30 µM) and agonist were added. After 10 min of incubation, the cells were spun down, the supernatant was removed, and the pellet was resuspended in 1 ml of 0.33 M perchloric acid containing about 2000 cpm [14C]cAMP. Cyclic AMP was isolated by sequential Dowex/alumina ion exchange chromatography (26), and radioactivity was determined in a liquid scintillation counter (Wallac, Turku, Finland). The conversion of [3H]ATP to [3H]cAMP was calculated as a percentage of total cellular [3H]ATP and normalized to the recovery of [14C]cAMP.

Receptor Binding Assay

Infected cells from monolayer cultures were harvested in phosphate-buffered saline solution and centrifuged 1500 × g for 5 min. The cell pellet was resuspended in cold potassium phosphate buffer (40 mM K2HPO4, 10 mM KH2PO4, pH7.4) and homogenized with an Ultra-Turrax homogenizer (Janke and Kunkel, Germany). 100-200 µg of protein of the homogenate was incubated with 10 nM [3H]RX821002 in a volume of 0.3 ml potassium phosphate buffer at 25 °C for 30 min. 10 µM phentolamine was used to determine nonspecific binding. The reactions were terminated by filtration through printed filtermat B filters (Wallac) using a Harvester 96 (Tomtec Inc., Orange, CO). After the filters had dried, a MeltiLex B/HS scintillator sheet was melted on them, and radioactivity was determined in a Microbeta scintillation counter (Wallac).


RESULTS

Expression of Mouse alpha 2-AR Subtypes

Infection of Sf9 cells with baculovirus harboring the genes for the mouse alpha 2A- or alpha 2B-AR resulted in a time-dependent increase in receptor density as determined by specific binding of [3H]RX821002 (data not shown). Cells infected with wild-type virus did not show specific binding of [3H]RX821002. When assayed for functional coupling to regulation of cAMP production, the alpha 2A subtype mainly inhibited the forskolin stimulation (Fig. 1A), whereas the alpha 2B subtype showed a biphasic response with inhibition at low concentration (1 µM) of norepinephrine (NE) and a potentiating effect at higher concentrations (100 µM) (Fig. 1B). The potentiation appeared to reach a maximum around 38 h postinfection (p.i.) and then to decline with longer infection times. For further characterization, 48 h p.i. was chosen because the magnitudes of inhibition versus potentiation of the forskolin response between the two subtypes were similar at this time point.


Fig. 1. alpha 2-AR responses in Sf9 cells at different time points postinfection. Sf9 cells were infected with recombinant baculovirus for different time periods, and changes in forskolin-stimulated cellular cAMP content were measured as described under "Experimental Procedures." Two different concentrations, 1 and 100 µM, of norepinephrine (NE) were used to show both the inhibition and potentiation with the alpha 2B subtype. The stimulation by forskolin alone is taken as 100%. The data are given as mean ± S.E. from two to four independent experiments performed in triplicate.
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In Fig. 2, the dose-response curves for the two receptor subtypes with two different agonists, NE and UK14,304, are shown. UK14,304 was used as a control agonist for inhibition since this compound has been shown to be very weak in potentiating forskolin stimulation with the alpha 2B subtype while being full agonist for the inhibition (27). Both NE and UK14,304 inhibited cAMP production in alpha 2A-expressing cells to the same extent and with similar potencies. In cells expressing the alpha 2B subtype, NE displayed a biphasic response with an inhibition at low concentrations, that reached a maximum at 1 µM, and that potentiated the forskolin stimulation at higher concentrations. UK14,304 elicited mainly inhibition with the alpha 2B subtype, confirming that UK14,304 is much less effective in eliciting a stimulation of cAMP production compared with the endogenous agonist NE.


Fig. 2. Dose-response profiles for alpha 2-ARs 48 h postinfection. Dose-response curves for two different agonists, norepinephrine (NE) and UK14,304 (UK), obtained with Sf9 cells expressing similar receptor levels of alpha 2-AR subtypes. The stimulation by forskolin alone is taken as 100%. The data points represent mean ± S.E. from two to four independent experiments performed in triplicate.
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Pertussis toxin (PTX) has been used in Chinese hamster ovary cells to abolish the inhibition of cAMP production through Gi proteins and thus reveal a less efficient coupling of alpha 2-ARs to a stimulatory component, presumably Gs (7, 19, 27). When Sf9 cells were treated with PTX for 48 h, the inhibition was abolished in both alpha 2A- and alpha 2B-expressing cells, and the stimulatory response with the alpha 2B subtype was enhanced over 2-fold (Fig. 3). Treatment of Sf9 cells with cholera toxin (CTX), which is known to activate Gs proteins persistently, drastically reduced the inhibitory response with both subtypes (Fig. 3).


Fig. 3. Effects of toxins and Ca2+ chelation on norepinephrine-induced cAMP production. Changes in cellular forskolin-stimulated cAMP levels were determined with 1 and 100 µM norepinephrine (NE) in Sf9 cells infected for 48 h with recombinant baculovirus for alpha 2A- or alpha 2B-AR. Pertussis toxin (PTX) (200 ng/ml) and cholera toxin (CTX) (10 µg/ml) were added at the time of infection. The measurements were performed as described under "Experimental Procedures" except for EGTA treatment where 0.5 mM EGTA was added to the assay buffer instead of 1 mM CaCl2. The data are expressed as percentage of forskolin-stimulated cAMP accumulation, with forskolin alone being 100%, and represent the mean ± S.D. from one to two independent experiments performed in triplicate.
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The influence of Ca2+ on the cAMP production was tested by chelating extracellular Ca2+ with EGTA. The chelation of extracellular Ca2+ did not effect the responses to any larger extent in cells expressing either the alpha 2A subtype or the alpha 2B subtype (Fig. 3).

Interchange of Third Intracellular Loop Does Not Affect Coupling Specificity

To try to delineate the domain in the alpha 2B subtype responsible for a potentiation of cAMP production, we expressed a set of chimeric alpha 2A-/alpha 2B-receptors (Fig. 4).


Fig. 4. Receptor constructs. Schematic illustration of the receptor constructs used to delineate the domain involved in subtype-specific coupling. The numbers in parentheses below the chimeric receptors denote the numbering of amino acids in the intact receptor subtype. For the point mutated receptors, the amino acid sequences of the second intracellular loops are lettered. Numbering of point mutations is for the alpha 2A subtype. Bmax values (mean ± S.D., n = 3) indicate the receptor levels in fmol/mg of protein as determined by specific binding of [3H]RX821002 to cell homogenate at 48 h postinfection unless otherwise noted.
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MCR1 and MCR2, alpha 2A and alpha 2B with interchange of the third intracellular loop (i3-loop), respectively, show similar dose-response curves with NE as the parent receptor (Fig. 5). MCR1 showed a reduced maximal inhibition compared with the alpha 2A subtype, but no biphasic response mode could be seen. MCR2 displayed a typical biphasic response but with a reduced potentiating effect. Employing UK14,304 as the agonist increased the maximal inhibition with MCR2 compared with the parent alpha 2B subtype, whereas the response with MCR1 did not deviate from the parent alpha 2A subtype (data not shown).


Fig. 5. Effects of i3-loop interchange. Dose-response curves for norepinephrine with chimeric receptors with i3-loop interchange between the alpha 2A- and alpha 2B-ARs. Sf9 cells were infected for 48 h with recombinant virus harboring the MCR1 or MCR2 constructs and cellular cAMP content was determined as described under "Experimental Procedures." The stimulation by forskolin alone is taken as 100%. The data points represent mean ± S.E. from two to three independent experiments performed in triplicate.
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MCR3, in which the carboxyl-terminal part from the end of the i3-loop of the alpha 2A subtype was exchanged with alpha 2B sequence, responded in the same way as the native alpha 2A subtype (Fig. 6). MCR4, with alpha 2B-sequence from the beginning of the second intracellular loop (i2-loop) to the C terminus, behaved as the intact alpha 2B subtype in displaying a biphasic dose-response curve with NE (Fig. 6), indicating that the second intracellular loop might be involved in the potentiation seen with the alpha 2B subtype.


Fig. 6. Effect of receptor-terminal sequences in determination of coupling specificity. Dose-response curves for norepinephrine obtained with Sf9 cells expressing MCR3 or MCR4 (see Fig. 4) 48 h postinfection. The stimulation by forskolin alone is taken as 100%. The data points represent mean ± S.E. from two independent experiments performed in triplicate.
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Effect of Second Intracellular Loop on Coupling Specificity

To test the involvement of the i2-loop in the different coupling modes, we expressed a chimeric alpha 2A receptor in which the i2-loop had been exchanged to alpha 2B receptor sequence, MCR5. This construct exhibited a similar biphasic dose-response curve with NE as the alpha 2B subtype 48 h p.i. (Fig. 7). Since this viral construct expressed receptors at somewhat higher density compared with the other constructs, we measured the dose-response relationship at 38 h p.i. when the receptor level was comparable to the parent alpha 2A subtype (see Fig. 4.). At 38 h p.i., this construct stimulated cAMP production very potently, showing almost no inhibition with NE (Fig. 7), which indicated that the change in coupling specificity could not be attributed to differences in the expression levels of the chimeric receptor.


Fig. 7. Effect of i2-loop substitution on coupling specificity. The i2-loop in the alpha 2A subtype was substituted for the equivalent sequence from the alpha 2B subtype. The dose-response curves for norepinephrine were obtained with Sf9 cells 38 h and 48 h postinfection. The data points represent mean ± S.E. from two independent experiments performed in triplicate.
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Site-directed Mutagenesis of the Second Intracellular Loop

There are six amino acid residues that differ between the alpha 2A and alpha 2B subtypes in the second intracellular loop (Fig. 4.). Three of these residues are essentially similar (Ile-135 in alpha 2A versus Val at the corresponding position in alpha 2B, Thr-136 versus Ser, and Ile-139 versus Leu). The three other non-conserved residues differ in terms of polarity (Ser-134 in alpha 2A versus Ala at the corresponding position in alpha 2B, Gln-137 versus Arg, and Leu-143 versus Ser). Site-directed mutagenesis of each of these three amino acids of the alpha 2A subtype to corresponding residues of the alpha 2B subtype did not give clear indications which residues might be responsible for the coupling specificity (Fig. 8.). These mutants all showed a lower degree of inhibition with NE compared with the alpha 2A subtype, and no marked biphasic response could be seen. On the contrary, a double mutant, S137A, L143S, exhibited a biphasic response with NE similar to the alpha 2B subtype although with a smaller magnitude of stimulation. All of the mutated constructs were expressed at receptor levels comparable with the alpha 2A subtype, and the inhibition with UK14,304 was 40-50% of forskolin stimulation with all four constructs (not shown).


Fig. 8. Coupling specificity of point mutants. Different point mutations were generated in the i2-loop of the alpha 2A-AR. The dose-response curves for norepinephrine were obtained with Sf9 cells 48 h postinfection. The data points represent mean ± S.E. from two independent experiments performed in triplicate.
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DISCUSSION

During the last few years, it has become evident that G protein-coupled receptors can couple to multiple G proteins to elicit different cellular responses (7, 28-30). The alpha 2-ARs have been shown to couple to both negative and positive regulation of adenylyl cyclase activity (5, 7, 9, 13-15). If coupling to both pathways occurs simultaneously but with different potencies, one could expect to obtain a biphasic dose-response curve. With the alpha 2-ARs, this is often the case; an inhibition of forskolin-stimulated cAMP production is seen with low concentrations of agonist and a stimulation or potentiation of cAMP production with higher concentrations (5, 7, 13, 15). Earlier studies have indicated that the stimulatory effect of alpha 2-ARs is cell-type-specific (15) and/or dependent on the expression level of the receptors (7). This response also seems to be subtype-specific, the alpha 2B subtype having a more pronounced stimulatory effect than the alpha 2A and alpha 2C subtypes when expressed in the same cells (8, 9, 13, 15, 31). In the present study, we expressed the mouse alpha 2A- and alpha 2B-AR subtypes in Sf9 cells at comparable expression levels and obtained a marked difference in the coupling specificity between the subtypes. The alpha 2B subtype showed a biphasic dose-response curve with a potentiation of the forskolin stimulation at high concentration of NE, whereas the alpha 2A subtype displayed a monophasic inhibitory dose-response curve. With prolonged infection times, which parallel an increase in receptor density, the maximal inhibition with alpha 2A was increased. This is in contrast to the finding by Eason et al. (7) that an increase in receptor density promotes the stimulatory pathway with the human alpha 2A subtype (alpha 2-C10). The reason for this discrepancy is unclear but might involve interspecies variation of the receptor subtypes or differences in the types of G proteins expressed by the two different cell lines used. A reduction in the maximal stimulation with longer infection times for the alpha 2B subtype was also seen, which indicates a more efficient coupling to the inhibitory component at higher expression levels. The imidazoline-like agonist UK14,304 was very weak in eliciting a biphasic response with the alpha 2B subtype. The ability of UK14,304 to promote coupling to a stimulatory pathway with the alpha 2B subtype has been shown to be very weak in Chinese hamster ovary cells (27).

PTX treatment, which is known to reveal a stimulatory pathway to cAMP production with the alpha 2-ARs (7, 19, 27), increased the stimulation over 2-fold with the alpha 2B subtype. This supports the hypothesis that coupling to both pathways occur simultaneously, and thereby, an elimination of either pathway would result in an enhancement of the other. PTX treatment of cells expressing the alpha 2A subtype did not reveal any significant coupling to a stimulatory pathway although the inhibition was abolished. This confirms that the ability of the receptors to potentiate cAMP production is, apart from being related to expression levels, also subtype-specific.

Treatment of the cells with CTX, which would abolish the stimulation if it was Gs-mediated, drastically reduced the inhibition with both subtypes but did not seem to affect the stimulatory component with the alpha 2B subtype. These data are difficult to interpret, however, since CTX stimulated the adenylyl cyclase activity several-fold over the forskolin-stimulated activity, and this activity may be difficult to inhibit (9, 32).

alpha 2-AR-mediated stimulation of adenylyl cyclase activity has been suggested to occur through a rise in intracellular calcium concentration in PC12 cells (15). Since we observed a small but significant elevation of intracellular Ca2+ in Sf9 cells expressing the alpha 2B subtype when assayed with fura-2 fluorescence (data not shown), we measured the change in cAMP production in the prescence of EGTA. The potentiating response did not differ significantly from the control experiment while EGTA largely prevented the Ca2+ elevation in the fura-2 assay. This suggests that Ca2+ elevation is not the stimulating factor in the alpha 2B-mediated potentiation of cAMP production in these cells although Ca2+ may enhance the stimulatory response.

The third intracellular loop of G protein-coupled receptors has been implicated in G protein selectivity and activation (for review, see Ref. 33). When we expressed chimeric alpha 2A-/alpha 2B-ARs with interchange of the i3-loops, the responses with NE were very similar to the parent receptor subtypes. A reduction of the potentiating response of alpha 2B was seen with MCR2, and a reduction of the inhibition of alpha 2A could be seen with MCR1. This may be related to a more efficient coupling to Gi proteins through the i3-loops of the receptors. Another possibility is that the change of the i3-loop might alter the general conformation of the coupling device leading to slightly altered responses.

In an earlier study, where part of the carboxyl-terminal tail of the alpha 2A subtype was introduced into the beta 2-AR, a small reduction in isoproterenol-stimulated adenylyl cyclase activity was seen, suggesting an involvement of the carboxyl-terminal tail in G protein coupling (21). In this study, the exchange of the carboxyl-terminal tail in alpha 2A subtype for alpha 2B sequence (MCR3) did not alter the coupling mode for the alpha 2A subtype (Fig. 6), indicating that this domain, if involved in coupling to G proteins in the alpha 2-ARs, probably interacts with a Gi protein. The chimeric receptor MCR4 displayed a typical biphasic dose-response curve. This was also expected since this chimera contains alpha 2B sequences in all the proposed G protein-coupling domains. We also constructed a chimeric alpha 2A receptor with alpha 2B sequence from the amino terminus to the distal end of the i2-loop, but this construct was not expressed at such a density that a functional characterization could be accomplished.

When the i2-loop from alpha 2B was introduced into the alpha 2A subtype, the chimeric receptor displayed a biphasic dose-response curve with NE similar to the alpha 2B subtype when assayed 48 h p.i. At an earlier time point (38 h p.i.), the chimeric receptor stimulated cAMP production even more potently than did the native alpha 2B subtype at the same time postinfection. There are two possible explanations for this. First, the chimeric construct was expressed at higher receptor levels than the alpha 2B construct, about 3.8 pmol/mg of protein for MCR5 compared with about 1.5 pmol/mg of protein for the alpha 2B subtype. A higher expression could increase coupling to the stimulatory component and thereby mask a coupling to the inhibitory component. Second, a Gs-coupling domain in the third intracellular loop of the human alpha 2A subtype has been identified based on studies with chimeric alpha 2/5-HT1A receptors (34). Exchange of the i2-loop from the alpha 2B subtype might result in a receptor that couples more tightly to a stimulating G protein.

The i2-loop has been implemented in G protein selectivity in a study using muscarinic m1:beta -adrenergic receptor chimeras (22). The role of the i2-loop in G protein activation has also been studied using synthetic peptides derived from alpha 2-ARs and muscarinic receptors (35, 36). In the study by Okamoto and Nishimoto (35), the peptide derived from the i2-loop of the human alpha 2A subtype (alpha 2-C10) potently stimulated GTPgamma S binding to Gs proteins. Although this finding is in contrast to our results that the alpha 2A receptor is strictly inhibiting, other regions of the receptor are probably also involved in governing the selectivity.

The i2-loop sequences of the alpha 2A and alpha 2B-ARs differ at six positions when predicted from the cDNA sequences. There are three non-conserved amino acid substitutions between the two subtypes that will change the polarity of the i2-loop, Ser-134, Gln-137, and Leu-143, in the alpha 2A subtype. Single point-mutated receptors with each of these non-conserved residues substituted with corresponding alpha 2B residues showed no clear biphasic responses with NE. In contrast, a double mutant with S134A and L143S responded in a similar way to the alpha 2B subtype. The interpretation of this is that at least these two residues are needed to maintain the integrity of the coupling domain. Interestingly, in the beta 2-adrenergic receptor, the equivalent residues (Ser-134 and Leu-143) are the same as in the alpha 2B subtype.

In conclusion, we have presented evidence that the coupling of alpha 2-ARs to cAMP production is subtype-specific and that the second intracellular loop of the alpha 2B subtype determines the different coupling specificity leading to stimulation of cAMP production. Site-directed mutagenesis of non-conserved amino acid residues indicates that the whole structure of the second intracellular loop is important for efficient coupling.


FOOTNOTES

*   This study was funded by the Technology Centre of Finland (TEKES), The Orion Corporation, The Waldemar von Frenckell Foundation, The Jusélius Foundation, The Ehrnrooth Foundation and The Borg Foundation.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: Dept. of Physiology and Medical Biophysics, Uppsala University, BMC, Box 572, S-75123 Uppsala, Sweden. Tel.: 46-18-174195 (direct); Fax: 46-18-174938.
1   The abbreviations used are: alpha 2-AR, alpha 2-adrenergic receptor; G protein, guanine nucleotide-binding protein; NE, norepinephrine; p.i., postinfection; PTX, pertussis toxin; CTX, cholera toxin; IBMX, isobutylmethylxanthine; MES, 4-morpholineethanesulfonic acid; MCR, mouse chimeric receptor; i2-loop, second intracellular loop.

ACKNOWLEDGEMENTS

We thank Dr. Brian Kobilka for providing the cDNA clones, Katariina Pohjanoksa for preparing the cDNA inserts, Kent Rönnholm for technical assistance, and Jyrki Kukkonen for constructive criticism.


REFERENCES

  1. Bylund, D. B., Ray-Prenger, C., and Murphy, T. J. (1988) J. Pharmacol. Exp. Ther. 245, 600-607 [Abstract]
  2. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., Francke, U., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1987) Science 238, 650-656 [Medline] [Order article via Infotrieve]
  3. Regan, J. W., Kobilka, T. S., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J., and Kobilka, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6301-6305 [Abstract]
  4. Lomasney, J. W., Lorenz, W., Allen, L. F., King, K., Regan, J. W., Yang-Feng, T. L., Caron, M. G., and Lefkowitz, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5094-5098 [Abstract]
  5. Fraser, C. M., Arakawa, S., McCombie, W. R., and Venter, J. C. (1989) J. Biol. Chem. 264, 11754-11761 [Abstract/Free Full Text]
  6. Cotecchia, S., Kobilka, B. K., Daniel, K. W., Nolan, R. D., Lapetina, E. Y., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1990) J. Biol. Chem. 265, 63-69 [Abstract/Free Full Text]
  7. Eason, M. G., Kurose, H., Holt, B. D., Raymond, J. R., and Liggett, S. B. (1992) J. Biol. Chem. 267, 15795-15801 [Abstract/Free Full Text]
  8. Jansson, C. C., Marjamäki, A., Luomala, K., Savola, J-M., Scheinin, M., and Åkerman, K. E. O. (1994) Eur. J. Pharmacol. 266, 165-174 [Medline] [Order article via Infotrieve]
  9. Jansson, C. C., Karp, M., Oker-Blom, C., Näsman, J., Savola, J-M., and Åkerman, K. E. O. (1995) Eur. J. Pharmacol. 290, 75-83 [CrossRef][Medline] [Order article via Infotrieve]
  10. Macnulty, E. E., McClue, S. J., Carr, I. C., Jess, T., Wakelam, M. J. O., and Milligan, G. (1992) J. Biol. Chem. 267, 2149-2156 [Abstract/Free Full Text]
  11. Michel, M. C., Brass, L. F., Williams, A., Bokoch, G. M., Lamorte, V. J., and Motulsky, H. J. (1989) J. Biol. Chem. 264, 4986-4991 [Abstract/Free Full Text]
  12. Kagaya, A., Mikuni, M., Yamamoto, H., Muraoka, S., Yamaki, S., and Takahashi, K. (1992) J. Neural. Transm. 88, 25-36 [Medline] [Order article via Infotrieve]
  13. Pepperl, D. J., and Regan, J. W. (1993) Mol. Pharmacol. 44, 802-809 [Abstract]
  14. Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R. (1992) Nature 356, 159-161 [CrossRef][Medline] [Order article via Infotrieve]
  15. Duzic, E., and Lanier, S. M. (1992) J. Biol. Chem. 267, 24045-24052 [Abstract/Free Full Text]
  16. Ullrich, S., and Wollheim, C. B. (1984) J. Biol. Chem. 259, 4111-4115 [Abstract/Free Full Text]
  17. Paris, H., Galitzky, J., and Senard, J. M. (1989) Mol. Pharmacol. 35, 345-354 [Abstract]
  18. Chabre, O., Conklin, B. R., Brandon, S., Bourne, H. R., and Limbird, L. E. (1994) J. Biol. Chem. 269, 5730-5734 [Abstract/Free Full Text]
  19. Jones, S. B., Halenda, S. P., and Bylund, D. B. (1991) Mol. Pharmacol. 39, 239-245 [Abstract]
  20. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1988) Science 240, 1310-1316 [Medline] [Order article via Infotrieve]
  21. Liggett, S. B., Caron, M. G., Lefkowitz, R. J., and Hnatowich, M. (1991) J. Biol. Chem. 266, 4816-4821 [Abstract/Free Full Text]
  22. Wong, S. K-F., Parker, E. M., and Ross, E. M. (1990) J. Biol. Chem. 265, 6219-6224 [Abstract/Free Full Text]
  23. Schneider, H., Feyen, J. H. M., and Seuwen, K. (1994) FEBS Lett. 351, 281-285 [CrossRef][Medline] [Order article via Infotrieve]
  24. Higushi, R. (1989) in PCR Technology (Erlich, H. A., ed), pp. 61-70, Stockton Press, New York
  25. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  26. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548 [Medline] [Order article via Infotrieve]
  27. Eason, M. G., Jacinto, M. T., and Liggett, S. B. (1994) Mol. Phamacol. 45, 696-702 [Abstract]
  28. Dell'Acqua, M. L., Carroll, R. C., and Peralta, E. G. (1993) J. Biol. Chem. 268, 5676-5685 [Abstract/Free Full Text]
  29. Prather, P. L., Loh, H. H., and Law, P. Y. (1994) Mol. Pharmacol. 45, 997-1003 [Abstract]
  30. Negishi, M., Irie, A., Sugimoto, Y., Namba, T., and Ichikawa, A. (1995) J. Biol. Chem. 270, 16122-16127 [Abstract/Free Full Text]
  31. Oker-Blom, C., Jansson, C., Karp, M., Lindqvist, C., Savola, J-M., Vlak, J., and Åkerman, K. (1993) Biochem. Biophys. Acta 1176, 269-275 [Medline] [Order article via Infotrieve]
  32. Dittman, A. H., Weber, J. P., Hinds, T. R., Choi, E-J., Migeon, J. C., Nathanson, N. M., and Storm, D. R. (1994) Biochemistry 33, 943-951 [Medline] [Order article via Infotrieve]
  33. Savarese, T. M., and Fraser, C. M. (1992) Biochem. J. 283, 101-119
  34. Eason, M. G., and Liggett, S. B. (1995) J. Biol. Chem. 270, 24753-24760 [Abstract/Free Full Text]
  35. Okamoto, T., and Nishimoto, I. (1992) J Biol. Chem. 267, 8342-8346 [Abstract/Free Full Text]
  36. McClue, S. J., Baron, B. M., and Harris, B. A. (1994) Eur. J. Pharmacol. 267, 185-193 [CrossRef][Medline] [Order article via Infotrieve]

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