©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a G(s) Coupling Domain in the Amino Terminus of the Third Intracellular Loop of the -Adrenergic Receptor
EVIDENCE FOR DISTINCT STRUCTURAL DETERMINANTS THAT CONFER G(s)VERSUS G(i) COUPLING (*)

(Received for publication, March 14, 1995; and in revised form, July 13, 1995)

Margaret G. Eason (1) Stephen B. Liggett (1) (2)(§)

From the  (1)Departments of Medicine (Pulmonary), (2)Molecular Genetics, and (3)Pharmacology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0564

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alpha(2)-Adrenergic receptors (alpha(2)AR) functionally couple not only to G(i) but also to G(s). We investigated the amino-terminal portion of the third intracellular loop of the human alphaAR (alpha(2)C10) for potential G(s) coupling domains using site-directed mutagenesis and recombinant expression in several different cell types. A deletion mutant and four chimeric receptors consisting of the alphaAR with the analogous sequence from the 5-HT receptor (a G(i)-coupled receptor) and the beta(2)AR (a G(s)-coupled receptor) were expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts, or COS-7 cells and examined for their ability to mediate stimulation or inhibition of membrane adenylyl cyclase activity or whole cell cAMP accumulation.

In stably expressing Chinese hamster ovary cells, deletion of amino acids 221-231, which are in close proximity to the fifth transmembrane domain, eliminated alpha(2)C10-mediated stimulation of adenylyl cyclase activity, while alpha(2)C10-mediated inhibition was only moderately affected. This suggested that this region is important for G(s) coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218-235 with 5-HT receptor sequence entirely ablated agonist-promoted G(s) coupling, as compared with a 338 ± 29% stimulation of adenylyl cyclase activity observed with the wild-type alpha(2)C10. In contrast, G(i) coupling for this mutant remained fully intact (57 ± 2% versus 52 ± 1% inhibition for wild-type alpha(2)C10). Similar substitution with beta(2)AR sequence had no effect on G(i) coupling but did reduce G(s) coupling. Two additional mutated alpha(2)C10 containing smaller substitutions of the amino-terminal region with 5-HT receptor sequence at residues 218-228 or 229-235 were then studied. While G(i) coupling remained intact with both mutants, G(s) coupling was ablated in the former but not the latter mutant receptor. Similar results were obtained using transfected Chinese hamster fibroblasts (which exclusively display alpha(2)AR-G(i) coupling) and COS-7 cells (which exclusively display alpha(2)AR-G(s) coupling). Thus, a critical determinant for G(s) coupling is contained within 11 amino acids(218-228) of the amino-terminal region of the third intracellular loop localized directly adjacent to the fifth transmembrane domain.

Taken together, these studies demonstrate the presence of a discrete structural determinant for agonist-promoted alpha(2)AR-G(s) coupling, which is distinct and separable from the structural requirements for alpha(2)AR-G(i) coupling.


INTRODUCTION

Activation of cellular signaling pathways by many hormones and neurotransmitters occurs via interaction with members of a superfamily of integral cellular membrane receptors that physically bind and activate heterotrimeric guanine nucleotide binding proteins (G-proteins). (^1)G-protein coupled receptors have an extracellular amino terminus and intracellular carboxyl terminus and are thought to span the cellular membrane seven times producing three extracellular and three intracellular loops. Chimeric receptor(1, 2, 3, 4, 5, 6) , site-directed mutagenesis(6, 7, 8, 9, 10, 11) , and peptide(12, 13, 14, 15, 16, 17) studies have clearly established that the G-protein coupling domains of these receptors are located within the intracellular portions, particularly in the third intracellular loop.

The adrenergic receptors (AR) mediate the effects of epinephrine and norepinephrine and are classified into several types: betaAR, alpha(1)AR, and alpha(2)AR. While the alpha(1)AR stimulate phosphatidylinositol hydrolysis via coupling to a G(q)/G class G-protein, the betaAR and alpha(2)AR are predominantly characterized by their abilities to modulate adenylyl cyclase activity. The betaAR are coupled to the stimulatory G-protein G(s), and thereby evoke stimulation of adenylyl cyclase activity resulting in the elevation of the intracellular second messenger cAMP. Conversely, the alpha(2)AR are primarily coupled to the inhibitory G-protein G(i), and in turn, inhibit adenylyl cyclase activity. There are numerous reports, however, that reveal that under certain circumstances, the alpha(2)AR elicit stimulation of cAMP production(18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) , and there are several lines of evidence that strongly suggest that alpha(2)AR are coupled directly to G(s) as well as G(i). Cerione et al.(30) demonstrated that purified human platelet alpha(2)AR (alphaAR) reconstituted in phospholipid vesicles with purified G(s) stimulates GTPase activity in an agonist-dependent manner. In our own work (21, 22) and that of others(23) , alpha(2)AR-mediated stimulation of adenylyl cyclase activity has been observed in assays using washed membrane preparations, thereby eliminating possible stimulatory signals from secondary downstream intracellular mediators that are independent of G(s) coupling. In addition, alphaAR-mediated stimulation of adenylyl cyclase activity in membrane preparations can be blocked both by cholera toxin (CTX) and by specific antiserum directed against G(21) . Finally, we have demonstrated physical alpha(2)AR-G(s) coupling using immunoprecipitation of solubilized agonist-alphaAR-G(s) complexes prepared from transfected Chinese hamster ovary (CHO) cells as identified by CTX-mediated ADP-ribosylation assays or Western blotting with G antiserum(21) .

Regardless of whether alphaAR-G(s) coupling is examined in intact cells or membranes, the process appears to be less efficient than G(i) coupling(18, 19, 20, 21, 22, 23, 24) . Typically, the agonist concentrations necessary to elicit detectable stimulation of adenylyl cyclase are higher than those for inhibition. Indeed, the EC values for agonist stimulation are 10-100-fold higher as compared with inhibition for alphaAR. In this regard, we have wondered if, in fact, alpha(2)AR contain distinct and separate structural determinants for coupling to G(s) or whether a recombinant system favors promiscuous association and activation of G(s) by predominantly G(i) coupling domains of the receptor. Delineating a specific region of the alphaAR, which confers agonist-mediated stimulation of adenylyl cyclase, would also provide additional confirmation that this signal is in fact the result of interaction at the level of the receptor itself rather than secondary effects due to other mechanisms evoked in the whole cell setting.

Indeed, most mutagenesis studies have been carried out with receptors that couple to a single G-protein. In some of these studies, the specificity of G-protein coupling has been attributed to the amino-terminal portion of the third intracellular loop. Interestingly, in previous studies, it has been shown that when this region of the alphaAR is substituted into the analogous position in the beta(2)AR that G(s) coupling is not affected(5, 6) , suggesting that the amino-terminal portion of the alphaAR can support G(s) coupling at least within the context of the beta(2)AR. This is supported by the studies of Okamoto and Nishimoto(15) , wherein a synthetic peptide based on this region of the alphaAR-stimulated GTPS binding to purified G(s)in vitro. Accordingly, in the present study, we have utilized deletion and chimeric mutagenesis of cloned human alphaAR (alpha(2)C10) to investigate the amino-terminal region of the third intracellular loop as a potential specific G(s) coupling domain.


EXPERIMENTAL PROCEDURES

Mutagenesis of alpha(2)C10

Construction of a deletion mutant and chimeric beta(2)AR/alpha(2)C10 or 5-HTR/alpha(2)C10 receptors was carried out using the human alpha(2)C10 cDNA construct in the mammalian expression vector pBC12BI(31) . These mutations are illustrated (see Fig. 1). First, a polymerase chain reaction technique was used to eliminate the nucleotides encoding amino acids 218-235 and to create a unique BspEI site corresponding to amino acids 217 and 236. Briefly, an oligonucleotide primer corresponding to a unique BglII restriction site was used in combination with a mutagenic primer encoding a new BspEI restriction site (located at amino acids 217-218) to allow for polymerase extension of a BglII/BspEI fragment. This fragment was digested with BglII and BspEI and ligated into digested alpha(2)AR-pBC12BI. Synthetic, complimentary oligonucleotides with overlapping ends cohesive to BspEI fragments, which encoded a short linker fragment to produce a mutant lacking amino acids 221-231 (DEL 221-231) or beta(2)AR or 5-HTR sequence analogous to amino acids 218-235 of alpha(2)C10, were phosphorylated, annealed, and then ligated into the above mutated alpha(2)C10-pBC12BI construct digested with BspEI. These two substitution mutants are denoted as alpha(2)(beta(2)) and alpha(2)(5-HT). Two additional mutations (see Fig. 1), which encoded for smaller substitutions of the 5-HTR sequence analogous to amino acids 218-228 and 229-235 (denoted as alpha(2)(5-HT 218-228) and alpha(2)(5-HT 229-235), respectively) were constructed in the same manner. All mutations were verified by dideoxy sequencing.


Figure 1: Schematic for deletion and substitution of amino-terminal residues in the third intracellular loop of alpha(2)C10. Shown is a schematic representation of the alpha(2)C10 with the indicated mutations. Substitution and deletion mutations were undertaken in the amino-terminal portion of the third intracellular loop, adjacent to transmembrane domain V (TM V). Amino acid alignment of this region for both wild-type and mutant alpha(2)C10 appears below. Underlined residues indicate wild-type alpha(2)C10 sequence. For the mutant denoted as DEL 221-231, amino acids 221-231 were deleted. This mutation resulted in the introduction of an alanine residue that is not present in wild-type alpha(2)C10. Mutants with substitution of amino acids 218-235 with the analogous sequences from the beta(2)AR or 5-HTR are denoted as alpha(2)(beta(2)) and alpha(2)(5-HT), respectively. Two additional mutants were also constructed that contain substitution of amino acids 218-228 and 229-235 with the respective sequence from the 5-HTR and are denoted as alpha(2)(5-HT 218-228) and alpha(2)(5-HT 229-235), respectively.



Cell Culture and Transfection

CHO cells and Chinese hamster fibroblasts (CHW) were grown in monolayers in Ham's F-12 media and Dulbecco's modified Eagle's media, respectively, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 80 µg/ml G418 (to maintain selection pressure) at 37 °C in a 5% CO(2) atmosphere. CHO and CHW cells were cotransfected with 30-40 µg of wild-type or mutant alpha(2)C10-pBC12BI and 3 µg of pSV(2)neo using a calcium phosphate precipitation method described by Cullen(32) . Stably expressing clones were identified using a [^3H]yohimbine binding assay as described below. COS-7 cells were grown in monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a 5% CO(2) atmosphere. For transient expression of wild-type and mutant alpha(2)AR, COS-7 cells in monolayers at 30-50% confluence were transfected with 10-15 µg of cDNA via a DEAE-dextran method as described previously(32) . COS-7 cells expressing wild-type and mutant alpha(2)AR were used for experiments on the second day following transfection. Mutant receptors DEL 221-231, alpha(2)(beta(2)), and alpha(2)(5-HT) were studied in CHO cells at expression levels of 10 pmol of receptor/mg of membrane protein along with wild-type alpha(2)C10 at a matched expression. Stable transfectants of mutant receptors alpha(2)(5-HT 218-228) and alpha(2)(5-HT 229-235) typically expressed levels of 5 pmol/mg of receptor in CHO cells and 2 pmol/mg of receptor in CHW cells and were studied in parallel with wild-type alpha(2)C10 expressing similar levels of receptor. For experiments, transfected cells in monolayers at 95% confluence were used, and multiple clones expressing mutant and wild-type alpha(2)C10 were studied. Permanent transfection of the cloned human beta(2)AR in pBC12BI into CHO cells was performed as above, and stably expressing clones were identified using a I-cyanopindolol binding assay exactly as described previously(33) . The CHO clonal cell line expressing the cloned human 5-HTR was obtained from John R. Raymond (Duke University) and is described elsewhere(34) .

Cholera and Pertussis Toxin Pretreatment and Membrane Preparation

For some experiments, alpha(2)AR-G(s) or G(i) coupling in CHO cells was isolated by pretreatment of cells with either CTX or pertussis toxin (PTX), respectively. Transfected CHO cells in monolayers were washed twice with phosphate-buffered saline and incubated in serum-free media containing either 20 µg/ml CTX or 500 ng/ml PTX for 24 h at 37 °C in a 5% CO(2) atmosphere. We have previously established that these concentrations of toxin eliminate agonist-promoted receptor-G(s) or -G(i) coupling, respectively, without being detrimental to the cells(21) . After toxin treatment, cells were washed 5 times with phosphate-buffered saline, and membranes were prepared by scraping with a rubber policeman in a hypotonic buffer (5 mM Tris-HCl, pH 7.4, 2 mM EDTA) followed by centrifugation at 40,000 times g for 10 min at 4 °C. Crude membrane pellets were then resuspended in the appropriate buffer for use in the assays below.

Adenylyl Cyclase Assay

CHO cells stably expressing mutant and wild-type alpha(2)C10 were untreated or exposed to CTX or PTX, membranes were prepared as described above, and then adenylyl cyclase activities were determined using a modification of the method of Salomon et al.(35) exactly as described previously(22) . Briefly, membranes (20 µg) were incubated with 2.7 mM phosphoenolpyruvate, 50 µM GTP, 0.1 mM cAMP, 0.12 mM ATP, 50 µg/ml myokinase, 0.05 mM ascorbic acid (to prevent oxidation of epinephrine), and 2.0 µCi of [alpha-P]ATP in a buffer containing 40 mM HEPES, pH 7.4, 25 mM NaCl, 1.6 mM MgCl(2), and 0.8 mM EDTA for 45 min at 37 °C. Reactions were terminated by the addition of a stop solution containing excess ATP and cAMP and 100,000 dpm of [^3H]cAMP. [P]cAMP and [^3H]cAMP were isolated by sequential chromatography over Dowex and alumina columns, and [^3H]cAMP was used to quantitate column recovery. Activities were measured in the presence of water (basal), 1.0 µM forskolin, and 1.0 µM forskolin with various concentrations of agonist. To assess the functional responses of beta(2)AR or 5-HTR expressed in CHO cells, transfected cells were incubated with vehicle (untreated) or the indicated toxin, membranes were prepared, and adenylyl cyclase activities were determined as described above, with the exception that forskolin was not included in assays of beta(2)AR-mediated stimulation.

Whole-cell cAMP Accumulation Assays

For some studies examining mutant and wild-type alpha(2)AR-G-protein coupling, accumulation of cAMP in intact cells was quantitated in stably expressing CHO and CHW cells or transiently expressing COS-7 cells. Briefly, cells expressing wild-type alpha(2)C10, or the mutants alpha(2)(5-HT 218-228) and alpha(2)(5-HT 229-235) growing in monolayers in multiwell (2 cm^2/well, 24 wells) plates, were washed twice with PBS and incubated in serum-free media containing 100 µM 3-isobutyl-1-methyl-xanthine (to inhibit phosphodiesterase activity) for 30 min at 37 °C in a 5% CO(2) atmosphere. Then, the indicated concentrations of agonist and, for experiments using CHO or CHW cells, 5 µM forskolin were added to duplicate wells. Incubations with agonist were carried out for 45 min (COS-7 cells) or 5 min (CHO and CHW cells), and reactions were stopped by the addition of 1 N HCl. Total cellular cAMP was quantitated using a radioimmunoassay as described previously(33) . Briefly, each sample and a series of cAMP standards were diluted up to 1.0 ml in 0.1 N HCl, and 40 µl of a 1:2.5 mixture of acetic anhydride/triethylamine was added. The resultant acetylated products were incubated with specific antisera for succinyl cAMP and 5 nCi of [I]cAMP tyrosine methyl ester. Reactions were passed over anion-exchange resin to clear the mixture of unbound cAMP, and the complexed [I]cAMP was eluted and counted in a counter.

Radioligand Binding Assays

Expression levels of mutant and wild-type alpha(2)C10 were determined using a [^3H]yohimbine binding assay as described previously(22) . Briefly, membranes prepared from cells expressing mutant or wild-type alpha(2)C10 were incubated in a Tris buffer (75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl(2), 2 mM EDTA) with 25 nM [^3H]yohimbine in the absence (total binding) and presence of 10 µM phentolamine (nonspecific binding) for 30 min at 37 °C. Specific binding was defined as the difference between total and nonspecific binding and was normalized for protein. For agonist competition studies, membranes were incubated in a buffer providing 50 mM Tris-HCl, pH 7.4, 10 mM MgSO(4), 0.5 mM EDTA with 6 nM [^3H]yohimbine, 100 µM GTP, and 12 different concentrations of the agonist epinephrine, ranging from 1 nM to 1 mM for 30 min at room temperature. All radioligand binding reactions were terminated by dilution with several volumes of ice-cold 10 mM Tris-HCl, pH 7.4, followed by rapid vacuum filtration through Whatman GF/C glass fiber filters.

Protein Measurement

Protein concentration was determined using the bicinchoninic acid method as described by Smith et al.(36) , with bovine serum albumin as standard.

Data Analysis

Adenylyl cyclase and cAMP accumulation dose-response data and radioligand binding data from competition experiments were analyzed by iterative least squares techniques(37) . For adenylyl cyclase and cAMP accumulation studies, data are reported as mean ± S.E. of the R(max) (maximal response) and the EC for epinephrine-mediated stimulation or inhibition from the indicated number of individual experiments. Comparisons for all experiments were made by two-tailed t tests, with significance imparted at p < 0.05.

Materials

[^3H]Yohimbine (80 Ci/mmol), [^3H]cAMP (31 Ci/mmol), [alpha-P]ATP (30 Ci/mmol), and I-cyanopindolol (2200 Ci/mmol) were from DuPont NEN. [I]cAMP tyrosine methyl ester (2200 Ci/mmol) was from Hazleton Washington (Vienna, VA). Forskolin, (-)-epinephrine, propranolol, and cholera toxin were from Sigma. Phentolamine was from Research Biochemicals. Pertussis toxin was from List Biologicals. Synthetic oligonucleotides were purchased from Oligos, Etc. (Wilsonville, OR). Geneticin (G418) was from Life Technologies, Inc. All tissue culture reagents were from JRH Biosciences. Other reagents were obtained from standard commercial sources.


RESULTS

To examine whether the amino-terminal region of the third intracellular loop of alpha(2)C10 possesses specific structural elements required for G(s) coupling, we constructed mutated alpha(2)C10 cDNAs, recombinantly expressed both mutant and wild-type genes in CHO cells, and assessed the ability of the expressed receptors to mediate stimulation and inhibition of adenylyl cyclase activity. The mutated receptors are illustrated in Fig. 1. The initial approach utilized a deletion mutant that lacked 10 amino-terminal residues of the third intracellular loop. Further studies were carried out using substitutions in this region with the analogous portions of other G-protein-coupled receptors in order to maintain a greater degree of overall structural integrity of the loop. Amino-terminal portions of the third intracellular loops of the G(s)-coupled beta(2)AR and the G(i)-coupled 5-HT receptor (5-HTR) were chosen since these receptors do not undergo dual G(s)/G(i) coupling. Mutant alpha(2)C10 containing 5-HTR or beta(2)AR substitutions for amino acids 218-235 are referred to as alpha(2)(5-HT) and alpha(2)(beta(2)), respectively. Two additional mutants contained smaller substitutions of amino acids at the most proximal(218-228) or distal(229-235) portions of this segment with the analogous sequence from the 5-HTR and are referred to as alpha(2)(5-HT 218-228) and alpha(2)(5-HT 229-235), respectively. All mutant alpha(2)C10 specifically bound the alpha(2)AR antagonist [^3H]yohimbine and displayed virtually identical affinities for the agonist epinephrine as compared with wild-type alpha(2)C10 (data not shown).

Initially, the involvement of the amino-terminal region in alpha(2)C10-G-protein coupling was explored using the mutant DEL 221-231. As shown in Fig. 2, the most striking result was found for G(s) coupling. For wild-type alpha(2)C10, following treatment with PTX, epinephrine-mediated stimulation of adenylyl cyclase activity was readily observable with a maximum stimulation of 338 ± 29% over forskolin-stimulated activity and an EC of 17 ± 1 µM. In contrast, under identical conditions, the mutant DEL 221-231 failed to stimulate adenylyl cyclase activity (Fig. 2). Following treatment of wild-type alpha(2)C10-expressing CHO cells with CTX, epinephrine-mediated inhibition of adenylyl cyclase activity was observed with a 52 ± 1% decrease from forskolin-stimulated activity and an EC of 151 ± 23 nM. For the mutant DEL 221-231, epinephrine-mediated inhibition was retained, although reduced as compared with wild-type alpha(2)C10, with a maximum inhibition of 30 ± 2% decrease from forskolin-stimulated activity, and a significantly greater EC of 19 ± 12 µM (p < 0.02 as compared with wild-type alpha(2)C10).


Figure 2: Deletion of residues 221-231 ablates alpha(2)C10-G(s) coupling. CHO cells stably expressing wild-type alpha(2)C10 and the mutant DEL 221-231 were pretreated with either CTX or PTX to isolate G(i) coupling or G(s) coupling, respectively, and adenylyl cyclase activities were measured in washed membranes as described under ``Experimental Procedures.'' Activities were determined in the presence of 1.0 µM forskolin and various concentrations of the agonist epinephrine. Results are expressed as the maximal percent change from forskolin-stimulated activity. Shown are mean ± S.E. from three experiments. *p < 0.02 as compared with wild-type alpha(2)C10.



As introduced earlier, subsequent studies were carried out utilizing 5-HTR/alpha(2)C10 and beta(2)AR/alpha(2)C10 chimeras, since we were concerned about potential nonspecific consequences of deletion mutations. To be certain that beta(2)AR and 5-HTR sequences were appropriate for such studies (i.e. that these receptors do not display dual G(s)/G(i) coupling), adenylyl cyclase studies were carried out using the beta(2)AR and 5-HTR permanently expressed in CHO cells under the same conditions as those used for the alpha(2)C10 mutants. These results are shown in Fig. 3. In membranes prepared from CHO cells stably expressing the beta(2)AR, the agonist isoproterenol elicited stimulation of adenylyl cyclase activity that was essentially eliminated (95% loss) following treatment with CTX. Note that following CTX, isoproterenol did not elicit beta(2)AR-mediated inhibition of adenylyl cyclase activity. Similarly, in membranes prepared from CHO cells expressing the 5-HTR, serotonin promoted inhibition of adenylyl cyclase activity that was entirely ablated following pretreatment of the cells with PTX. After PTX, agonist-promoted stimulation of adenylyl cyclase activity was not detected with the 5-HTR (Fig. 3).


Figure 3: beta(2)AR-G(s) coupling and 5-HTR-G(i) coupling. CHO cells expressing beta(2)AR or 5-HTR were incubated in the absence (UNTREATED) or presence of either CTX or PTX to ablate beta(2)AR-G(s) coupling or 5-HTR-G(i) coupling, respectively. Membranes were prepared, and adenylyl cyclase activities were determined in the presence of the indicated concentrations of the beta(2)AR agonist isoproterenol or the 5-HTR agonist serotonin. Shown are results from a single experiment representative of four performed. The functional responses of the beta(2)AR and 5-HTR were entirely eliminated by pretreatment with CTX and PTX, respectively, which demonstrates that these two receptors are not dually coupled to G(i) and G(s).



CHO cells permanently expressing alpha(2)(beta(2)), alpha(2)(5-HT), and wild-type alpha(2)C10 were exposed to CTX and PTX to dissect the G(i)- and G(s)-coupling pathways, respectively. Then, washed membranes were prepared, and adenylyl cyclase activities were determined in the presence of the agonist epinephrine as before. Substitution of the amino-terminal portion of the third intracellular loop with 5-HTR sequence resulted in a complete loss of alpha(2)C10-mediated stimulation of adenylyl cyclase activity (Fig. 4). In contrast, G(i) coupling remained intact and displayed the wild-type phenotype with a 57 ± 2% decrease in adenylyl cyclase activity and an EC for epinephrine-mediated inhibition of 220 ± 3 nM (p is not significant as compared with wild-type alpha(2)C10, Fig. 4). The results with the mutant consisting of substituted beta(2)AR sequence supported the above concept that the amino terminus of the third intracellular loop of alpha(2)C10 is critical for G(s) coupling. The alpha(2)(beta(2)) mutant did display G(s) coupling, although the maximal response was diminished by 75% as compared with wild-type alpha(2)C10. Similar to what was found above, beta(2)AR substitution did not reduce G(i) coupling; in fact, the maximum inhibition was slightly greater than that of wild-type alpha(2)C10 (67 ± 2 versus 52 ± 1% decrease in adenylyl cyclase activity, respectively, p < 0.05, Fig. 4). Thus, the preservation of G(i) coupling with both mutations suggests that the loss of G(s) coupling observed with substitution of this region is due to loss of a specific G(s) coupling domain.


Figure 4: Effects of substitution of amino acids 218-235 on alpha(2)C10-G(i) and -G(s) coupling. CHO cells expressing wild-type alpha(2)C10 and the mutants alpha(2)(beta(2)) and alpha(2)(5-HT) were incubated with either CTX (closed symbols) or PTX (open symbols) to isolate G(i) or G(s) coupling, respectively. Squares, wild-type alpha(2)C10; diamonds, alpha(2)(beta(2)); circles, alpha(2)(5-HT). Adenylyl cyclase activities were determined in washed membranes in the presence of 1.0 µM forskolin and the indicated concentrations of the agonist epinephrine. Results are expressed as the percent of forskolin-stimulated activity. Shown are the mean ± S.E. from three to five experiments.



Inasmuch as G(s) coupling was completely removed by substitution of amino acids 218-235 with 5-HTR sequence, we concluded that the key residues within this region that are critical for alpha(2)C10-G(s) coupling are contained within these 18 amino acids. Interestingly, the first 11 residues(218-228) of this 18-amino acid sequence are relatively conserved (>80%) among the three human alpha(2)AR subtypes, while there are virtually no identities among the next 7 amino acids(229-235). Hence, we considered whether the requirements for alpha(2)C10-G(s) coupling are contained within these 11 amino acids and represent a G(s) coupling domain that is conserved among all alpha(2)AR. In this regard, we constructed two additional mutants containing smaller substitutions with 5-HTR sequence within this 18 amino acid region (Fig. 1). One mutant, termed alpha(2)(5-HT 218-228), contained substitution of the first 11 amino acids with the analogous sequence from the 5-HTR. The other mutant, termed alpha(2)(5-HT 229-235), contained substitution of only the last seven amino acids within this region with the analogous 5-HTR sequence.

CHO cells expressing matched expression levels of the mutants alpha(2)(5-HT 218-228) and alpha(2)(5-HT 229-235) and wild-type alpha(2)C10 were studied under the same conditions as before. As shown in Fig. 5, in membranes expressing wild-type alpha(2)C10, epinephrine elicited stimulation of adenylyl cyclase activity with a maximum stimulation of 192 ± 18% of forskolin-stimulated activity and an EC of 16 ± 0.4 µM. Similarly, for the mutant alpha(2)(5-HT 229-235), epinephrine-mediated stimulation occurred with a maximum stimulation of 185 ± 7% of forskolin-stimulated activity and an EC of 24 ± 3 µM. In contrast, the mutant alpha(2)(5-HT 218-228) appeared not to couple to G(s) in that no epinephrine-mediated stimulation of adenylyl cyclase activity was detected (Fig. 5). As with previous mutations, both substitutions had no effect on alpha(2)C10-G(i) coupling. Wild type alpha(2)C10 displayed epinephrine-mediated inhibition with a 52 ± 3% decrease in adenylyl cyclase activity and an EC of 182 ± 26 nM. For alpha(2)(5-HT 218-228) and alpha(2)(5-HT 229-235), epinephrine-mediated inhibition of adenylyl cyclase was virtually identical to wild-type alpha(2)C10 with a maximum inhibition of 49 ± 3% and 56 ± 5% decrease from forskolin-stimulated activity, respectively, and ECs of 151 ± 10 nM and 83 ± 12 nM, respectively. These data, with the maximal G(i) or G(s) responses normalized to wild-type alpha(2)C10 for all the mutations, are summarized in Table 1.


Figure 5: alpha(2)(5-HT 218-228), alpha(2)(5-HT 229-235), and wild-type alpha(2)C10-G(s) coupling. Adenylyl cyclase activities were determined in membranes prepared from PTX-treated CHO cells expressing wild-type alpha(2)C10 and the mutants alpha(2)(5-HT 218-228) and alpha(2)(5-HT 229-235). Activities were measured in the presence of 1.0 µM forskolin and the indicated concentrations of epinephrine. Shown are the mean ± S.E. from three to four experiments.





In additional experiments, we also assessed both membrane adenylyl cyclase assays and cAMP accumulation studies in intact CHO cells expressing wild-type alpha(2)C10 and the mutant alpha(2)(5-HT 218-228), which had not been pretreated with either CTX or PTX. Shown in Fig. 6A are the results of membrane adenylyl cyclase assays in the presence of the agonist epinephrine. As we have previously reported(21) , without pretreatment with either toxin, alpha(2)C10-mediated modulation of adenylyl cyclase activity in CHO cells was complex and biphasic, consisting of both an inhibitory (G(i) coupling) and stimulatory (G(s) coupling) component (Fig. 6A). In contrast, the mutant alpha(2)(5-HT 218-228) displayed only monophasic inhibition, revealing a loss of G(s) coupling but not G(i) coupling. In complimentary studies assessing whole-cell cAMP accumulation, similar results were obtained. Wild-type alpha(2)C10 mediated a biphasic cAMP accumulation response, while a predominantly inhibitory response was found with the mutant alpha(2)(5-HT 218-228) (Fig. 6B).


Figure 6: Membrane adenylyl cyclase and whole-cell cAMP accumulation with wild-type and mutant alpha(2)C10 in CHO cells without pretreatment with toxin. A, adenylyl cyclase activities were determined in membranes prepared from CHO cells expressing wild-type alpha(2)C10 and the mutant alpha(2)(5-HT 218-228) in the presence of 1.0 µM forskolin and the indicated concentrations of epinephrine. B, cAMP accumulation in intact CHO cells expressing wild-type alpha(2)C10 and the mutant alpha(2)(5-HT 218-228) was determined as described under ``Experimental Procedures.'' Shown are the mean ± S.E. from four to five experiments. Absent error bars denote standard errors that were obscured by the size of the symbol and were < 5%.



Finally, we examined the ability of wild-type alpha(2)C10 and the mutant alpha(2)(5-HT 218-228) to couple to both G(i) and G(s) using two transfected cell lines in which alpha(2)C10-modulation of adenylyl cyclase activity has been observed to be either exclusively inhibitory (CHW cells) or stimulatory (COS-7 cells). For these studies, intact cAMP accumulation studies were performed in the presence of the specific alpha(2)AR-agonist UK-14304. In COS-7 cells transiently expressing wild-type alpha(2)C10, UK-14304 elicited a 10-fold stimulation of cAMP accumulation with an EC of 319 ± 74 nM (Fig. 7). Conversely, the mutant alpha(2)(5-HT 218-228) in COS-7 cells displayed no detectable stimulation of cAMP (Fig. 7). As was found above, substitution of amino acids 218-228 did not reduce alpha(2)AR-G(i) coupling. In CHW cells, both wild-type alpha(2)C10 and alpha(2)(5-HT 218-228) inhibited adenylyl cyclase activity similarly with EC values for agonist-mediated inhibition of 5.6 ± 1.7 versus 3.6 ± 0.9 nM, respectively, and R(max) values of 69.2 ± 1.9 versus 85.5 ± 1.7% decrease in forskolin-stimulated activity, respectively (Fig. 7).


Figure 7: Whole-cell cAMP accumulation in COS-7 and CHW cells expressing wild-type alpha(2)C10 and the mutant alpha(2)(5-HT 218-228). Whole-cell cAMP accumulation studies were performed in the presence of the indicated concentrations of the alpha(2)AR agonist UK-1304 using COS-7 or CHW cells expressing wild-type alpha(2)C10 and the mutant alpha(2)(5-HT 218-228) as described under ``Experimental Procedures.'' For observation of alpha(2)AR-mediated inhibition in CHW cells, 1.0 µM forskolin was included in the assay. Shown are the mean ± S.E. from four experiments. Absent error bars denote standard errors that were obscured by the size of the symbol and were < 5%.




DISCUSSION

The seemingly paradoxical ability of alpha(2)AR to mediate stimulation of cAMP production has been reported in pancreatic islet cells(28) , cerebral cortical brain slices(29) , and a number of recombinantly expressing clonal cell lines including CHO cells(18, 19, 21, 22, 23) , COS-7 cells(20) , HEK-293 cells(20, 24) , PC-12 cells(25) , JEG-3 cells(26) , and the S115 mouse mammary tumor cell line(27) . Although the underlying mechanism for these alpha(2)AR-mediated increases in cAMP has been a matter of some debate, recent studies, as outlined earlier (see the Introduction), have established compelling evidence that alpha(2)AR directly couple to G(s) and thereby elicit stimulation of adenylyl cyclase activity. While coupling to multiple signaling transduction pathways is not uncommon among G-protein-coupled receptors, the ability of alpha(2)AR to couple to G(s) as well as to G(i) is particularly intriguing in that, as such, the alpha(2)AR are capable of simultaneously evoking both stimulatory and inhibitory regulation of the activity of a single effector. The primary aim of the current work was to explore the nature of alpha(2)AR-G(s) coupling to determine if, indeed, alpha(2)AR contain specific structural elements for G(s)versus G(i) coupling. Based on studies of other G-protein coupled receptors(3, 4, 5, 6, 7, 8, 9, 10, 12) , in vitro peptide studies (15) , and the results from our initial deletion mutation (Fig. 2), we focused on the amino-terminal region of the third intracellular loop of alpha(2)C10 as a potentially selective G(s) coupling domain.

In early studies, we found that deletion of amino acids 221-231 entirely ablated alpha(2)C10-mediated stimulation of adenylyl cyclase activity, yet alpha(2)C10-mediated inhibition was still present, albeit somewhat diminished and less efficient as compared with wild-type alpha(2)C10 (Fig. 2). In order to further determine the specificity of these effects on functional alpha(2)C10-G-protein coupling with less drastic mutations than such a deletion, we constructed and assessed the coupling characteristics of a series of chimeric 5-HTR/alpha(2)C10 and beta(2)AR/alpha(2)C10 receptors (Fig. 1). With this approach, the amino-terminal region was replaced with sequence from the beta(2)AR, which only couples to G(s), and sequence from the 5-HTR, which only couples to G(i) (Fig. 3). Thus, with the beta(2)AR substitution, we could discern losses in alpha(2)C10-G(i) coupling, and conversely, with 5-HTR substitution we could ascertain losses in alpha(2)C10-G(s) coupling. We found that substitution of amino acids 218-235 with the analogous 5-HTR sequence entirely removed the ability to elicit epinephrine-mediated stimulation of adenylyl cyclase activity (Fig. 4). Smaller substitutions with the analogous 5-HTR sequence further revealed that the necessary requirements for alpha(2)C10-G(s) coupling in this region are confined to a small stretch of 11 amino acids (RIYQIAKRRTR) directly adjacent to the fifth transmembrane domain (Fig. 5Fig. 6Fig. 7). This loss of functional alpha(2)C10-G(s) coupling appears to be due to removal of specific G(s) coupling domains, in that the G(i)-coupled pathway remained fully intact with both 5-HTR and beta(2)AR substitutions (Table 1, Fig. 4, Fig. 6, and Fig. 7).

Although no studies have explored G(s) coupling domains of alpha(2)AR, there are several reports that support our findings. In intact receptor studies, O'Dowd et al.(6) and Liggett et al.(5) have reported that substitution of the amino-terminal domain of the third intracellular loop of the beta(2)AR with the analogous sequence from the alpha(2)C10 has very little, if any, effect on beta(2)AR-mediated stimulation of adenylyl cyclase activity. Moreover, consistent with our current results that this region is not critical for alpha(2)C10-G(i) coupling, alpha(2)C10 substitution in this region of the beta(2)AR does not confer G(i) coupling to the beta(2)AR(5) . Interestingly, unlike what is found with alpha(2)AR substitution of this region in the beta(2)AR, we found that substitution of this region in alpha(2)C10 with beta(2)AR sequence reduces alpha(2)C10-G(s) coupling (Fig. 4). One possible explanation for this is that the amino-terminal domain of the beta(2)AR, when placed into the context of an alpha(2)AR, is not sufficient to promote full functional G(s) coupling. Also consistent with our current result (although not in the context of an intact receptor), a synthetic peptide, which corresponds to the sequence RIYQIAKRRTR in alpha(2)C10, has been found to directly activate purified G(s)in vitro(15) .

For a number of G-protein coupled receptors, the amino-terminal domain of the third intracellular loop has been found to play an important role in receptor-G-protein coupling(3, 4, 7, 8, 9, 10, 11, 13, 15, 16) . For the muscarinic acetylcholine receptors (4, 8, 11) and the turkey betaAR (9) this region has been proposed to determine the specificity of these receptors coupling to their respective G-proteins. It should be noted that similar sequences to the RIYQIAKRRTR in alpha(2)C10 are found in the analogous region of the other two human alpha(2)AR subtypes alpha(2)C4 (RIYRVAKRRTR) and alpha(2)C2 (RIYLIAKRSNR), and, as we have previously shown(21) , all three alpha(2)AR subtypes share the ability to bind and activate G(s). Therefore, it is likely that this region serves as a critical component in G(s) coupling for alpha(2)C4 and alpha(2)C2 as well. Still, while it is clear that this sequence is important for functional alpha(2)C10-G(s) coupling, it is important to consider that it may not be the sole determinant. For the beta(2)AR, which has been the most extensively studied, it is becoming evident that multiple intracellular domains are required for binding and activation of G(s)(5, 6, 7, 10, 13) . Thus, in addition to this region there may be other key regions within the alpha(2)AR that contribute to the process of G(s) coupling and stimulation.

A large number of G-protein-coupled receptors have been found to exert multiple intracellular effects, whether through coupling to multiple G-proteins, through activation of multiple effectors by an individual G-protein, or via the secondary influences of one signaling cascade on others. For example, some receptors can stimulate phosphoinositide turnover and inhibit cAMP formation, while others have been reported to stimulate both phosphoinositide turnover and cAMP formation. In the latter case, for the rat thyrotropin receptor (38) and rat neurotensin receptor(39) , mutagenesis studies have delineated structural elements within the intracellular domains that selectively support stimulation of cAMP and/or phospholipase C activation. Thus, in agreement with our findings for alpha(2)C10, the divergent coupling pathways of other G-protein-coupled receptors appear to have separable structural requirements. The novelty of the current study lies in both the unique ability of alpha(2)AR to dually couple to G(i) and G(s) and the first definition of specific elements within a G(i)-linked receptor that confer G(s) coupling.

Although the physiological significance of G(s) activation by alpha(2)AR remains unclear, there are now multiple cell lines and tissues, as stated above, in which this aspect of alpha(2)AR signaling has been observed. As can be noted in intact cell cAMP studies ( Fig. 7and (22) ), the stimulation response, although less efficient than the inhibitory response, nevertheless occurs with an EC that is submicromolar, which may be in the physiologic range. Also, there is growing evidence that the alpha(2)AR may not be the only class of G(i)-coupled receptors that can produce stimulation of cAMP. For example, the m(4)-muscarinic receptor, when expressed in CHO cells, elicits biphasic modulation of cAMP formation with a stimulatory component that is not Ca-dependent and is not blocked by PTX(40) . In a recent report, m(4)-muscarinic modulation of cAMP formation was examined in HEK-293 cells co-expressing type I and III adenylyl cyclases(41) . In these studies, the m(4) receptor displayed stimulation of cAMP that was Ca-independent, GTP-dependent, and insensitive to PTX, supporting a direct G(s) mechanism. The opioid receptor endogenously expressed in a neuroblastoma cell line produces stimulation of basal adenylyl cyclase activity, which is blocked by CTX but not by PTX(42) . Also, the cloned human and dog 5-HT receptors expressed in CHO cells and Y1 Kin-8 cells, respectively, exert stimulatory effects on cAMP accumulation that has been attributed to G(s) coupling(43) . Functional coupling to the stimulation of cAMP generation appears to be specific to certain G(i) coupled receptors in that there are examples of inhibitory receptors that display solely inhibition of adenylyl cyclase activity, even in CHO cells that appear to be a useful system in discerning dual G(i)/G(s) coupling. Examples include the human 5-HT ( (44) and Fig. 3), the rat µ opioid receptor(45) , and somatostatin receptor subtypes 1, 2, and 5(46, 47) .

In conclusion, we report that amino acids 218-228 in the amino-terminal region of the third intracellular loop of alpha(2)C10 constitute a discrete structural domain required for functional alpha(2)C10-G(s) coupling. Moreover, this sequence is not required for functional alpha(2)C10-G(i) coupling and thus represents a selective G(s) coupling domain that is distinct and separable from the requirements for G(i) coupling. Thus, it appears that alpha(2)AR have evolved to possess specific structural determinants that confer the ability of these receptors to couple to two G-proteins with opposing actions on a single effector enzyme.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL41496 and HL53436 (to S. B. L.) and a Department of Defense National Defense Science and Engineering Graduate Fellowship (to M. G. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: 231 Bethesda Ave., Rm. 7511, P. O. Box 670564, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835.

(^1)
The abbreviations used are: G-protein, guanine nucleotide binding protein; AR, adrenergic receptors; CHO, Chinese hamster ovary; CTX, cholera toxin; PTX, pertussis toxin; CHW, Chinese hamster fibroblasts; alpha(2)C10, alpha(2)C4, and alpha(2)C2, alpha(2)AR subtypes localized to human chromosomes 10, 4, and 2, respectively; 5-HTR, serotonin type 1A receptor; G(i), inhibitory G-protein; G(s), stimulatory G-protein; COS-7 cells, SV40 transformed African Green monkey kidney cells.


ACKNOWLEDGEMENTS

We thank Kenneth J. Oswald and Elizabeth T. Donnelly for technical assistance and Cheryl T. Theiss for tissue culture and transfection.


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