INTRODUCTION

Adrenergic receptors (AR)¹ are members of a superfamily of integral membrane proteins that signal to the interior of the cell through heterotrimeric guanine nucleotide binding proteins or G-proteins. The AR are divided into three classes, ₁, ₂, and , which are differentiated by their relative selectivity for certain ligands, signal transduction pathways, and molecular structure. Each class of AR has been further divided into several pharmacological subtypes, each of which represents distinct receptors, as demonstrated by the cloning and recombinant expression of their respective genes from a variety of species, including human. There are three cloned human ₂AR, ₂C10, ₂C4, and ₂C2, that correspond to the pharmacologically defined subtypes _2A, _2C, and _2B, respectively (1, 2, 3). The ₂AR display a wide distribution in both peripheral (4, 5) and central (5, 6, 7) tissues and mediate a variety of physiological responses (8).

The ₂AR primarily couple to the inhibitory G-protein G_i, which negatively modulates the activity of the enzyme adenylyl cyclase in the production of the second messenger cAMP. In addition to the inhibition of adenylyl cyclase activity, ₂AR also have been linked to a number of other cellular signaling pathways via coupling to G_i or a G_i/G_o class G-protein (9), activation of phospholipase C activity (10) (potentially via coupling to a G_q/G₁₁ class protein (11)), and even stimulation of adenylyl cyclase activity via coupling to G_s (12, 13). With respect to the latter signaling pathway, increases in cAMP in response to ₂AR agonists have been observed in cerebral cortical brain slices (14) and pancreatic islet cells (15), as well as a host of transfected cell lines including CHO cells (12, 16, 17, 18, 19, 20), COS-7 cells (13, 18, 20), HEK-293 cells (13), PC-12 cells (21), JEG-3 cells (22), and the S115 mouse mammary tumor cell line (23). In many of these cell lines, at least for the _2AAR, both receptor-mediated inhibition and stimulation of cAMP production can be readily observed and occur as a complex, biphasic response. In this regard, we have utilized CHO cells as a model system to evaluate and compare ₂AR-mediated inhibition and stimulation within a single cell type. This has proved particularly useful in some of our most recent studies in which the ability of mutated ₂AR to couple to G_i and G_s was examined (20).

Site-directed mutagenesis, chimeric receptor, and in vitro peptide studies have indicated that the intracellular portions, particularly the second and third intracellular loops, of G-protein-coupled receptors are the regions that physically interact with G-proteins. For the ₂AR, very little is known concerning the regions within the intracellular domains of these receptors that functionally interact with G-proteins. In some of the earliest mutagenesis studies, substitution of ₂AR sequence into the ₂AR was utilized to elucidate regions within the ₂AR required for G_s coupling (24, 25, 26); however, no information concerning the specific regions involved in ₂AR-G-protein coupling was obtained. Furthermore, subsequent to the above studies, an ₂AR-G_s coupling pathway was delineated (12). In other studies, it has been shown that synthetic peptides based on relatively small portions of the intracellular regions of the human _2AAR activate purified G-proteins in vitro (27, 28). And, in a competition peptide study, incubation of human platelet membranes with synthetic peptides based on the second intracellular loop and the carboxyl-terminal region of the third intracellular loop of the human _2AAR reduced high affinity agonist binding (29). None of these studies, however, directly identified a region or regions within the intact ₂AR that are required for functional G-protein coupling.

Recently, using deletion and chimeric mutagenesis of the cloned human _2AAR, we identified a discrete stretch of 11 amino acids (218-228) in the amino-terminal region of the third intracellular loop that is required for functional G_s coupling but not for G_i coupling (20). This provided evidence that there are specific structural domains within ₂AR that enable the receptor to couple to G_s, which are distinct and separable from the structural requirements for ₂AR-G_i coupling. In the present study we have explored the second and third intracellular loop regions of the _2AAR to delineate domains responsible for functional G_i coupling. We have also further assessed potential requirements for G_s coupling using an expanded array of chimeric receptors. And finally, when several critical sequences were found, we considered whether they represent redundant, fully competent domains within the _2AAR for coupling to a single G-protein or whether multiple domains act together to accomplish functional coupling. Studies were undertaken using a series of nine chimeric receptors in which portions of the _2AAR were substituted with the analogous sequence from either the G_s-coupled ₂AR or the G_i-coupled serotonin type 1A receptor (5-HT_1AR). Mutant and wild type ₂AR were stably expressed in CHO cells, and agonist-mediated inhibition and stimulation of adenylyl cyclase activity was assessed in membrane assays.

EXPERIMENTAL PROCEDURES

The cloned human _2AAR (₂C10) in the mammalian expression vector pBC12BI was used as a template for mutagenesis (1). For mutagenesis, several regions of the _2AAR were substituted with analogous sequence from either the cloned human 5-HT_1AR or ₂AR. These regions included the amino- and carboxyl-terminal portions of the third intracellular loop and the second intracellular loop (see Fig. 1). Substitution of amino acids 218-235 with analogous ₂AR and 5-HT_1AR sequence in the amino-terminal region of the third intracellular loop has been previously described (20). For substitutions in the carboxyl-terminal region of the third intracellular loop, a BglII/KpnI fragment of ₂C10-pBC12BI was subcloned into M13mp18, and then NheI and Mlu I restriction sites were created at positions corresponding to amino acids 352-353 and 375-376, respectively, using oligonucleotide-directed mutagenesis (30). The fragment containing the new restriction sites was ligated back into digested ₂C10-pBC12BI to create a cassette for substitution. Then ₂C10-pBC12BI containing sites for NheI and Mlu I was digested with these enzymes and then ligated with annealed, synthetic oligonucleotides that encoded either ₂AR or 5-HT_1AR sequence analogous to amino acids 355-371 of ₂C10. For second intracellular loop substitutions, a SacI fragment of ₂C10-pBC12BI was subcloned into M13mp19. Oligonucleotides encoding sequence for the analogous ₂AR or 5-HT_1AR second loop sequence with 5 and 3 tags containing complementary ₂C10 sequence directly carboxyl- and amino-terminal to the substitution were utilized to simultaneously remove and replace amino acids 133-149 of ₂C10. Mutated fragments were then ligated back into digested ₂C10-pBC12BI. Combinations of the above substitutions were constructed by splicing together restriction digest fragments containing the desired mutations. All mutations were verified by dideoxy sequencing.

Targeting of potential G-protein coupling domains of the human

AR using substitution with analogous sequences from the human

CHO cells were grown in monolayers in Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin at 37 °C in a 5% CO₂ atmosphere. For stable expression of mutant and wild type ₂AR, CHO cells at ~30% confluence were cotransfected using calcium phosphate precipitation (31) with 30 µg of wild type or mutant cDNA in pBC12BI and 3 µg of pSV₂neo, which provides for G418 resistance. Transfected cells were isolated in medium containing 1 mg/ml G418. G418-resistant cells were expanded, and confluent monolayers were screened for expression of ₂AR using a [³H]yohimbine binding assay as described below. Selection pressure for clonal cell lines was maintained by the addition of 80 µg/ml of G418 to the above medium. For all studies, transfected cells in monolayers at ~90% confluence were utilized, and multiple clones expressing each receptor were studied.

For membrane studies, cells in monolayers were rinsed three times with phosphate-buffered saline, and membranes were prepared by scraping with a rubber policeman in ice-cold hypotonic lysis buffer (5 mM Tris-HCl, 2 mM EDTA, pH 7.4) followed by centrifugation at 42,000 × g for 10 min at 4 °C. Crude membrane pellets were then resuspended in the appropriate buffer for use in the assays. Protein concentration in prepared membranes was measured using the copper-bicinchoninic acid method (32) with bovine serum albumin as standard.

Expression of mutant and wild type ₂AR was determined using a [³H]yohimbine binding assays as described previously (12). Briefly, membranes from stably expressing CHO cells prepared as described above were incubated with 25 nM [³H]yohimbine in a 75 mM Tris-HCl, 12.5 mM MgCl₂, 2 mM EDTA, pH 7.4 buffer in the absence or the presence of 100 µM phentolamine for 30 min at 37 °C. Specific binding was normalized for protein concentration. Saturation binding studies were carried out by incubation of membranes with various concentrations of [³H]yohimbine ranging from 0.5 to 30 nM in the absence and the presence of 10 µM phentolamine in the above buffer for 30 min at 37 °C. For agonist competition studies, membranes were incubated in 50 mM Tris-HCl, 10 mM MgSO₄, 0.5 mM EDTA, pH 7.6 with ~6 nM of [³H]yohimbine with 12 concentrations of ()-epinephrine ranging from 0.1 nM to 1 mM in the absence or the presence of 100 µM GTP for 30 min at 37 °C. Reactions were terminated by dilution with several volumes of an ice-cold 10 mM Tris-HCl pH 7.4 buffer and vacuum filtration through Whatman GF/C glass fiber filters.

₂AR-mediated inhibition or stimulation was determined in membranes prepared from CHO cells stably expressing mutant and wild type ₂AR using the method of Salomon et al. (33) as modified (12). Previously, we have shown that ₂AR-mediated responses in CHO cells are biphasic, consisting of both inhibitory and stimulatory components, due to the ability of ₂AR to couple to both G_i and G_s (12). In order to isolate either monophasic ₂AR-mediated inhibition or stimulation, transfected CHO cells were pretreated with either 20 µg/ml of CTX or 500 ng/ml of PTX, respectively, for 16 h prior to membrane preparation and adenylyl cyclase assays. In all experiments, mutant and wild type ₂AR were studied at matched expression levels ranging from ~5-10 pmol of receptor/mg of membrane protein. Activities were determined in the presence of water (basal), 1.0 µM forskolin, and 1.0 µM forskolin with the indicated concentrations of the ₂AR agonist epinephrine.

Data from adenylyl cyclase and agonist competition radioligand binding assays were analyzed by iterative least squares techniques (34). For determination of whether agonist competition data were best fit by a simple model (one-site curve) or a more complex model (two-site curve), F-test analysis was used with assignment of the more complex model when p < 0.05.

[³H]Yohimbine (80 Ci/mmol), [-³²P]ATP (30 Ci/mmol), and [³H]cAMP (31 Ci/mmol) were from DuPont NEN. Cholera toxin, forskolin, and epinephrine were from Sigma. Pertussis toxin was from List Biologicals. Geneticin (G418), penicillin, and streptomycin were from Life Technologies, Inc. Ham's F-12 medium and all other tissue culture reagents were from JRH Biosciences.

RESULTS

Shown in Fig. 1 are the regions of the intracellular domains of the _2AAR that were investigated as potential G_i or G_s coupling domains. Three principal regions were chosen based on studies with other G-protein-coupled receptors, primarily the ₂AR (24, 25, 26, 35, 36, 37, 38), and the muscarinic receptors (39, 40), and studies using synthetic peptides based on regions of the human _2AAR (27, 28, 29). These included the second intracellular loop and the most amino- and carboxyl-terminal regions of the third intracellular loop region directly adjacent to transmembrane domains V and VI, respectively. For mutagenesis, each region of the _2AAR was substituted with the analogous sequence from either the cloned human ₂AR or 5-HT_1AR. Due to the coupling pathways of the ₂AR (G_s-coupled) and the 5-HT_1AR (G_i-coupled), mutant ₂AR containing substitution with ₂AR sequence were primarily utilized to discern losses in ₂AR-G_i coupling, and mutants containing substitution with 5-HT_1AR sequence were primarily utilized to discern losses in ₂AR-G_s coupling. CHO cells were stably transfected with the cDNAs encoding mutant and wild type ₂AR, and then pharmacological characterization and functional adenylyl cyclase assays were performed in washed membranes to determine the consequences of these mutations.

All the mutant ₂AR bound the antagonist [³H]yohimbine with high affinity and displayed guanine nucleotide-sensitive high affinity agonist binding (Table I). This suggests that the mutations made did not result in global conformational changes affecting ligand binding, which might confound subsequent interpretation of functional responses. Some small but statistically significant differences in K_i values were found with some of the mutants as compared with the wild type _2AAR, as indicated (Table I). There were also some differences in K_L or K_H values; however, the relevance of such changes in the absence of functional studies was not clear at this juncture. Receptor expression for the wild type _2AAR was 7 ± 1 pmol/mg of membrane protein (n = 11), and for each experiment, mutant and wild type ₂AR were studied at matched levels of receptor expression.

Table I. Pharmacological characterization of mutant and wild type 2 AR receptors Shown are the results from [3H]yohimbine saturation and ()-epinephrine competition binding assays performed using mutant and wild type 2AR as described under ``Experimental Procedures.'' The results from [3H]yohimbine saturation experiments are expressed as the mean of two independent experiments, each performed in duplicate, and the results from competition binding assays are expressed as the mean ± S.E. of three to four experiments performed. Kd, equilibrium dissociation constant for [3H]yohimbine; Ki, equilibrium dissociation constant for epinephrine in the presence of 100 µM GTP; KH and KL, high and low affinity equilibrium dissociation constants for epinephrine in the absence of GTP; RH, percentage of receptors occupying the high affinity binding state for epinephrine in the absence of GTP. Shown are the results from [3H]yohimbine saturation and ()-epinephrine competition binding assays performed using mutant and wild type 2AR as described under ``Experimental Procedures.'' The results from [3H]yohimbine saturation experiments are expressed as the mean of two independent experiments, each performed in duplicate, and the results from competition binding assays are expressed as the mean ± S.E. of three to four experiments performed. Kd, equilibrium dissociation constant for [3H]yohimbine; Ki, equilibrium dissociation constant for epinephrine in the presence of 100 µM GTP; KH and KL, high and low affinity equilibrium dissociation constants for epinephrine in the absence of GTP; RH, percentage of receptors occupying the high affinity binding state for epinephrine in the absence of GTP. Receptor Substitution (amino acids) [3H]Yohimbine Kd Epinephrine Ki (GTP) KL KH RH nM µM µM nM %  2AAR 2.4 1.1  ± 0.1 1.4  ± 0.3 6.7  ± 1.8 52  ± 5  2(2 2L) 133 -149 2.6 4.7  ± 0.3a 1.2  ± 0.2 14.6  ± 3.2 45  ± 1  2(5-HT 2L) 133 -149 2.7 0.5  ± 0.03a 0.5  ± 0.1a 12.8  ± 1.8 58  ± 2  2(2 NT) 218 -235 4.7 1.3  ± 0.1 1.1  ± 0.2 11.2  ± 2.3 38  ± 1  2(5-HT NT) 218 -235 3.8 0.8  ± 0.1 0.6  ± 0.1 4.4  ± 1.6 51  ± 2  2(2 CT) 355 -371 3.2 0.3  ± 0.05a 0.8  ± 0.1 7.6  ± 1.4 64  ± 3  2(5-HT CT) 355 -371 3.2 1.2  ± 0.3 1.9  ± 0.2 6.4  ± 2.4 44  ± 2  2(2 NT + CT) 218 -235 2.7 1.5  ± 0.2 2.6  ± 0.9 127  ± 44.7a 56  ± 5 355 -371  2(5-HT NT + CT) 218 -235 3.1 1.6  ± 0.2 1.7  ± 0.7 8.1  ± 3.5 69  ± 5 355 -371  2(2 CT + 2L) 355 -371 2.6 2.7  ± 0.3a 2.5  ± 0.9 40.1  ± 5.2a 47  ± 2 133 -149 a p < 0.02 as compared with the wild type 2AAR.

In order to isolate and study ₂AR-G_i versus G_s coupling in membrane adenylyl cyclase assays, transfected CHO cells were pretreated with either CTX or PTX, respectively, as described under ``Experimental Procedures.'' Previously, we have extensively confirmed that losses in functional G-protein coupling observed using membranes prepared from transfected CHO cells pretreated with toxin are also found using untreated CHO cells (seen as a loss of one limb of the biphasic response) and using cell lines that exclusively display either ₂AR-mediated inhibition or stimulation of adenylyl cyclase activity (20). Therefore, in order to assess the loss of one or both of these coupling pathways in a single cell system and to simplify analysis with monophasic dose-response curves for inhibition or stimulation, we have utilized transfected CHO cells pretreated with the appropriate toxin.

Shown in Fig. 2 and summarized in Table II are the results from substitution in the amino and carboxyl-terminal regions of the third intracellular loop. Following treatment of CHO cells expressing the wild type _2AAR with PTX, epinephrine elicited an increase of 308 ± 25% over forskolin-stimulated adenylyl cyclase activity with an EC₅₀ of 16 ± 1 µM (Fig. 2A and Table II). Following treatment with CTX, epinephrine elicited a decrease of 53 ± 2% in forskolin-stimulated adenylyl cyclase activity with an EC₅₀ of 183 ± 11 nM (Fig. 2B and Table II). In assays performed with the mutant ₂(5-HT NT) under the same conditions, no epinephrine-mediated stimulation was observed (Fig. 2A), whereas epinephrine-mediated inhibition occurred identically to that observed for the wild type _2AAR (Fig. 2B). Similarly, the mutant ₂(₂ NT) elicited epinephrine-mediated inhibition of adenylyl cyclase activity to the same extent as the wild type receptor with no loss in the maximum inhibition (67 ± 2% versus 53 ± 2% of forskolin-stimulated activity, respectively) and only a slight increase in the EC₅₀ (490 ± 60 nM versus 183 ± 11 nM, respectively). Thus, we considered the loss in the stimulatory pathway to be due to specific removal of G_s coupling determinants in that ₂AR-G_i coupling remained entirely intact. Interestingly, although ₂AR substitutions were not primarily used to observe losses in ₂AR-G_s coupling and despite the importance of the amino-terminal domain of the ₂AR in ₂AR-G_s coupling (35, 36, 37), the maximum epinephrine-mediated stimulation found with the mutant ₂(₂ NT) was reduced by ~75% with a ~2.5-fold increase in the EC₅₀ as compared with the wild type receptor (Fig. 2A and Table II). Taken together, these results indicate that the amino-terminal domain of the third intracellular loop is absolutely required for wild type functional ₂AR-G_s coupling but not for ₂AR-G_i coupling. Also, the data suggest that other regions of the receptor support G_i coupling, because this function was not altered with the ₂AR substitution.

Effects of substitution in the amino- and carboxyl-terminal regions of the third intracellular loop on

Table II. Summary of EC50 and Rmax values for agonist-mediated inhibition and stimulation of adenylyl cyclase activity with mutant and wild type 2AR Summarized are the results from membrane adenylyl cyclase experiments using mutant and wild type 2AR as described in Figs. 3, 4, 5. The results are expressed as the mean ± S.E. Rmax values for inhibition and stimulation of adenylyl cyclase activity are expressed as the maximal percentage of decrease in forskolin-stimulated activity and the maximal percentage of increase over forskolin-stimulated activity, respectively. ND, not determined; NA, not applicable. Summarized are the results from membrane adenylyl cyclase experiments using mutant and wild type 2AR as described in Figs. 3, 4, 5. The results are expressed as the mean ± S.E. Rmax values for inhibition and stimulation of adenylyl cyclase activity are expressed as the maximal percentage of decrease in forskolin-stimulated activity and the maximal percentage of increase over forskolin-stimulated activity, respectively. ND, not determined; NA, not applicable. Receptor Gi coupling Gs coupling Rmax EC50 Rmax EC50 % decrease nM % increase µM  2AAR 53  ± 2 183  ± 11 308  ± 25 16  ± 1  2(2 2L) 35  ± 3a 4920  ± 1330a 489  ± 21a 20  ± 2  2(5-HT 2L) 47  ± 3 160  ± 60 -0- NA  2(2 NT)b 67  ± 2a 490  ± 60a 83  ± 6a 40  ± 5a  2(5-HT NT)b 57  ± 2 220  ± 3 -0- -0-  2(2 CT) 42  ± 1a 120  ± 20 270  ± 24 6  ± 1  2(5-HT CT) 56  ± 4 280  ± 40 36  ± 5a 19  ± 3  2(2 NT + CT) 46  ± 3 11700  ± 100a -0- NA  2(5-HT NT + CT) 67  ± 2a 280  ± 80 ND ND  2(2 CT + 2L) 42  ± 6 295000  ± 79400a 466  ± 60a 3  ± 0.2a a p < 0.02 as compared with the wild type 2AAR. b Data published previously (20).

Similar to what was found with substitution in the amino-terminal region, the mutant ₂(5-HT CT) displayed markedly reduced functional ₂AR-G_s coupling as compared with the wild type _2AAR with a 36 ± 5% versus 308 ± 25% increase over forskolin-stimulated adenylyl cyclase activity, respectively (Fig. 2A). Again, these losses appeared to be specific in that epinephrine-mediated inhibition was fully intact with this mutation (Fig. 2B and Table II). For ₂(₂ CT), epinephrine-mediated inhibition occurred with no change in the EC₅₀ and, although statistically significant, only a slight reduction in the maximum decrease in forskolin-stimulated adenylyl cyclase activity (42 ± 1% versus 53 ± 2% with wild type _2AAR). Thus, even with substitution of the ₂AR sequence in the carboxyl terminus of the third intracellular loop, the ₂AR functionally couples to G_i with the wild type phenotype. This was also what was found when ₂AR sequence was substituted in the amino terminus of the third intracellular loop. That is, ₂AR-G_i coupling was not affected. This suggested that either these two regions are not important in G_i coupling or that perhaps either domain of the _2AAR could independently provide for functional coupling to G_i. On the other hand, G_s coupling was ablated by the 5-HT_1AR substitution in the carboxyl terminus. Because this also occurred with 5-HT_1AR substitution in the amino terminus, these results suggested that both regions are required for G_s coupling and do not function independently. Finally, substitution of the carboxyl-terminal region with ₂AR sequence entirely supported ₂AR-G_s coupling, which was not an unexpected finding because this region of the ₂AR has been shown to be a G_s coupling domain (25, 26, 36, 38).

The second intracellular loop was found to be necessary for both the stimulatory and inhibitory pathways. For the mutant ₂(₂ 2L), a substantial loss in epinephrine-mediated inhibition was observed with a ~27-fold increase in the EC₅₀ and a ~34% reduction in the maximum inhibition as compared with the wild type _2AAR (Fig. 3A and Table II). For the mutant ₂(5-HT 2L), epinephrine-mediated stimulation was entirely absent (Fig. 3B and Table II). Interestingly, for the mutant ₂(5-HT 2L) epinephrine-mediated inhibition occurred to a similar extent as the wild type receptor (Fig. 3A). The mutant ₂(₂ 2L) did not display a loss of ₂AR-mediated stimulation; in fact, epinephrine-mediated stimulation was augmented as compared with the wild type _2AAR (Fig. 3B). These latter data suggest that the second intracellular loop regions of the 5-HT_1AR and the ₂AR are capable of specifically supporting their respective pathways, inhibition and stimulation of adenylyl cyclase activity, within the context of the _2AAR. By inference, within the second intracellular loop of the wild type _2AAR, the necessary elements for both pathways are present.

Effects of substitution in the second intracellular loop on

Because the single ₂AR substitutions in the third intracellular loop had little or no effect on G_i coupling and substitution in the second intracellular loop regions did not entirely eliminate it, we constructed and expressed two combinations of the original ₂AR mutants to examine the effects of substitution of multiple regions on ₂AR-mediated inhibition of adenylyl cyclase activity: one in which both the amino- and carboxyl-terminal regions of the third intracellular loop were substituted with the analogous sequences from the ₂AR, referred to as ₂(₂ NT + CT) and one in which the carboxyl-terminal region of the third intracellular loop and the second intracellular loop were substituted with the analogous sequences from the ₂AR, referred to as ₂(₂ CT + 2L). The results from adenylyl cyclase assays performed on CTX-pretreated membranes from CHO cells expressing ₂(₂ NT + CT) and ₂(₂ CT + 2L) as well as those from the single ₂AR substitution mutations are shown in Fig. 4A. For the mutant ₂(₂ NT + CT), epinephrine-mediated inhibition was significantly reduced with a ~64-fold increase in the EC₅₀ as compared with the wild type _2AAR (Fig. 4A and Table II). This loss in ₂AR-mediated inhibition was most likely due to removal of specific G-protein coupling domains in that similar studies using a complementary mutant in which these two regions were substituted with 5-HT_1AR sequence (₂(5-HT NT + CT)) demonstrated full preservation of the inhibitory pathway (Table II). Thus, although individual substitution of either the amino- or carboxyl-terminal side of the third intracellular loop does not affect functional ₂AR-G_i coupling, substitution of both sides substantially reduced G_i coupling. These findings suggest that both of the third intracellular loop regions are important components in ₂AR-G_i coupling; however, it appears that each region can efficiently serve to fully support functional G_i coupling domain in the absence of the other.

Effects of combined

AR substitutions on

Because the ₂(₂ 2L) mutant displayed a loss of G_i coupling, we wondered whether a combination mutation that included one of the third intracellular loop regions would be even more dysfunctional. This turned out to be the case, because the mutant ₂(₂ CT + 2L) showed the most profound loss in ₂AR-G_i coupling with a ~1600-fold increase in the EC₅₀ for epinephrine-mediated inhibition of forskolin-stimulated adenylyl cyclase activity as compared with the wild type receptor (Fig. 4A and Table II). In fact, no epinephrine-mediated inhibition of adenylyl cyclase activity was detected with this mutant except in the presence of the highest agonist concentrations in the assay. Thus, if the degree of agonist-mediated inhibition is compared between all of the ₂AR-substituted mutations at a single concentration of agonist (30 µM) that elicits maximum inhibition with the wild type _2AAR, the pattern shown in Fig. 4B becomes evident. As shown, individual substitution of either the amino- or carboxyl-terminal regions of the third intracellular loop only minimally affected, if at all, the level of ₂AR-mediated inhibition. Equally significant losses in G_i coupling are found with substitution of the second intracellular loop, alone, or substitution of both of the third intracellular loop regions together (>80% loss in the extent of inhibition of adenylyl cyclase activity as compared with the wild type _2AAR). Substitution of the second intracellular loop and one of the carboxyl-terminal side of the third intracellular loop together further reduces functional ₂AR-G_i coupling to a complete loss of agonist-promoted inhibition of adenylyl cyclase activity at this concentration (Fig. 4B).

DISCUSSION

These studies have elucidated several key regions of the _2AAR required for functional G-protein coupling. Although a number of mutagenesis studies have been carried out with G_s-coupled receptors (24, 25, 26, 35, 37, 38, 41, 42, 43, 44, 45), only a few have been reported with G_i-coupled receptors (39, 40, 46), and these have been limited in scope. None of these studies have examined coupling domains within a receptor, such as the ₂AR, that is dually coupled to G_s and G_i. For the second intracellular loop, we found that substitution with ₂AR sequence markedly perturbed G_i coupling, and substitution with 5-HT_1AR sequence similarly affected G_s coupling. Thus, within the second intracellular loop of the wild type _2AAR, domains supportive of both G_s and G_i coupling are present. This is consistent with studies of other G-protein-coupled receptors that indicate a critical role of the second intracellular loop for G-protein coupling/specificity (44, 45), although its necessity for both an efficient (G_i) and a somewhat less efficient (G_s) pathway in a dually coupled receptor could not be predicted from such studies.

Within the third intracellular loop, we found evidence for two G_i coupling domains, one in the amino terminus and the other in the carboxyl terminus. When one of these regions is removed by substitution with ₂AR, the mutant receptor appears to be minimally affected in its coupling efficiency to G_i. When both are substituted, G_i coupling is markedly impaired. This suggests that there are redundant, fully competent G_i coupling domains within the ₂AR third intracellular loop. An alternative explanation would be that the ₂AR sequences used for substitution support, to some extent, G_i coupling. We do not believe that this is tenable based on several observations. First, we have recently shown (20) that a deletion _2AAR mutant, where 11 amino acids of the amino terminus of the third loop were deleted, still had the capacity to couple to G_i, presumably via the other domain in the carboxyl terminus. Secondly, several of the ₂AR substitution mutants reported here displayed efficient coupling to G_i (equivalent to the wild type _2AAR), which would imply that the substituted sequences, if they support G_i coupling, are highly efficient in doing so. Studies with wild type and mutant ₂AR have not, however, indicated any detectable G_i coupling with wild type receptor (20, 26). An entirely different set of findings were obtained when G_s coupling was examined. For this pathway, substitution of 5-HT_1AR sequence in either the amino or carboxyl terminus resulted in a loss of G_s coupling. This implies, in contrast to what was found for G_i coupling, that G_s coupling requires domains within both the amino and carboxyl terminus. Redundant G_s coupling domains within the _2AAR, then, appear unlikely.

There are some notable differences between our results and those of Okamoto and Nishimoto where synthetic peptides based on _2AAR sequences corresponding to amino acids 131-148 (second intracellular loop region), 218-229 (amino-terminal region of the third intracellular loop), and 356-371 (carboxyl-terminal region of the third intracellular loop) were studied for their ability to stimulate GTPS binding to the purified G-proteins G_i, G_o, and G_s in vitro (27, 28). Each peptide displayed a specific pattern of GTPS stimulation with each of the G-proteins examined. The peptide representing the second intracellular loop of the _2AAR specifically stimulated GTPS binding to G_s with high efficacy but did not stimulate GTPS binding to G_i or G_o. The peptide representing amino-terminal domain of the third intracellular loop stimulated binding to all three G-proteins similarly with low efficacy, whereas the peptide representing the carboxyl-terminal domain stimulated binding to G_i and G_o with similar efficacies and to G_s with a significantly lower efficacy. Our findings with the intact receptor provide strong support for a role of the second intracellular loop in ₂AR-G_s coupling. However, in contrast to the peptide-based studies, we found that this region is also critically involved in functional ₂AR-G_i coupling as well (Figs. 3 and 4 and Table II). Furthermore, despite the low efficacy of GTPS binding reported for the amino- and carboxyl-terminal peptides for G_s coupling, we found that these regions, like the second intracellular loop, were both critically important for functional stimulation of adenylyl cyclase activity by ₂AR. In agreement with the peptide studies, our data demonstrate that both the amino and carboxyl terminus of the third intracellular loop support ₂AR-G_i coupling.

The most intriguing finding in our studies is that either the amino or the carboxyl terminus of the third intracellular loop can be substituted and the inhibitory pathway remains intact, yet clearly these regions are important in ₂AR-G_i coupling because dual substitution greatly impairs the inhibitory response as does substitution of one of these regions in addition to the second intracellular loop. As mentioned above, this could perhaps be explained by the presence of structural elements within both regions that can equally support coupling to G_i, thereby endowing the ₂AR with redundant G_i coupling domains. That there may be redundant coupling domains to a single G-protein within a receptor has not been previously proposed. There are potential advantages for a receptor to have evolved in this manner. In the case of the ₂AR, redundant domains for G_i coupling might serve the maintain a certain degree of function despite regulatory processes that occur via covalent modification of the receptor by kinase-mediated phosphorylation and/or association with regulatory molecules. That is, the extent of desensitization may be limited by the presence of two G_i coupling domains. Although it is difficult to make comparisons, this notion is supported by the fact that agonist-promoted desensitization of recombinantly expressed ₂AR (47) is greater than that observed with the _2AAR expressed in the same cell line (48). Also, redundant domains may serve as a protective mechanism in the case of mutagenic events, which might introduce detrimental changes in one or the other region. It is also possible that structural features within these two domains of the ₂AR may also allow for the receptor to simultaneously couple to similar G-proteins, such as the separate G_i isoforms. This cannot be addressed in the current study, because CHO cells express predominantly G_i3 (49).

In summary, these studies have provided several novel insights into the structural elements that govern functional ₂AR-G-protein coupling. We have identified three key domains involved in both G_i and G_s coupling of ₂AR, which include the second intracellular loop and the amino- and carboxyl-terminal regions of the third intracellular loop. In addition, our results suggest that these regions serve distinct roles for G_i versus G_s coupling. For G_s coupling, each of these regions is absolutely required. In contrast, for G_i coupling, the second intracellular loop is absolutely required in addition to one of either the amino- or carboxyl-terminal region of the third intracellular loop. Each of the latter two regions appears to be capable of fully supporting ₂AR-G_i coupling in the absence of the other and, in this manner, represent redundant G-protein coupling domains, each of which can independently function along with the second intracellular loop to provide for ₂AR-G_i coupling.