Introduction

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₁- and ₃-AR are the predominant -AR subtypes in adipocytes, and analysis of these subtypes has revealed several important pharmacological and biochemical differences [for review, see Granneman (1995)]. For example, catecholamines, the natural ligands of these receptors, exhibit a much higher binding affinity for ₁-AR. ₃-AR have a relatively low affinity for catecholamines, yet that low affinity is largely compensated by a much higher degree of coupling efficiency. Thus, ₃-AR fully activate adenylyl cyclase when relatively few receptors are occupied, as indicated by the large difference between binding K_d and EC₅₀ values for cAMP accumulation.

In addition, ₃-AR, which are highly expressed in adipose tissues, have received considerable attention as a target for antiobesity and antidiabetes therapeutics (Granneman, 1995). In this regard, several substituted phenethanolamines have been described that potently activate the ₃-AR, yet have little or no activity at ₁-AR. These agents have proven to be very effective in animal models of obesity and diabetes, owing to their action on adipocyte ₃-AR (Bloom et al., 1992; Susulic et al., 1995; Grujic et al., 1997).

The structural bases for these characteristic pharmacological differences are not completely known. Chimeric receptors have been used successfully to explore the structure/function relationships among adrenergic receptor subtypes. Previous analyses of the ₃-AR subtype have used chimeras with ₂-AR and have focused largely upon analysis of receptor desensitization and sequestration (Liggett et al., 1993; Nantel et al., 1993; Jockers et al., 1996). Although certain differences between the "atypical" ₃-AR and the "typical" ₁- and ₂-AR have been noted (Granneman, 1992; Liggett et al., 1993; Nantel et al., 1993; Chaudhry and Granneman, 1994; Jockers et al., 1996), certain functional differences among these subtypes are unique or more robust between ₁- and ₃-AR subtypes. Therefore, we have performed direct comparisons between rat ₁- and ₃-AR and among chimeric and mutated receptors composed of these subtypes. These experiments have focused upon characteristic pharmacological properties of rat ₁- and ₃-AR subtypes, including catecholamine binding affinity, coupling to cAMP generation, and interaction with ₃-AR-selective phenethanolamines.

	Materials and Methods

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Construction of chimeric and mutated receptors. The cloning of the rat ₁- and ₃-AR cDNAs has been described (Granneman et al., 1991, Chaudhry and Granneman, 1992). The first chimera was constructed using the technique of Moore and Blakely (1994). This construct (₁/₃-O3) contained amino acids encoded by the ₁ receptor to Val334, at the end of TM region 6, followed by ₃-AR sequence beginning at Leu311. The complementary chimera (₃/₁-O3) was made by polymerase chain reaction mutagenesis and encoded the ₃-AR to Pro317, followed by ₁-AR sequence beginning at Arg339. Chimeras that substituted a region from TM5 through TM6 (including the third intracellular loop, I3) were constructed from the above chimeras using the common BsmBI site to replace codons before ₁-AR Ser232 in TM5 with ₃-AR sequence to form ₃(₁TM5-6), and ₁ sequence to ₃-AR Ser209 to form ₁(₃TM5-6). The TM5-6 chimeras also contained a binding site for the M2 monoclonal antibody (IBI) on the amino terminus of the receptors. Epitope tagging had no effect on the pharmacological properties of native rat ₁- and ₃-AR. For the final construct, Phe350 and Phe351 of the ₁-AR were changed to alanine and leucine found in the ₃-AR by replacing the HindIII to BglI fragment with a synthetic double-stranded oligonucleotide encoding the desired mutation to form ₁-F350A,F351L. All mutations and amplified sequences were verified by dideoxynucleotide sequencing.

Mammalian cell expression. Constructs were cloned into the mammalian expression vector pRc/CMV or the closely related derivative pcDNA3 (Invitrogen). These vectors contain the cytomegalovirus promoter to drive expression and the neomycin resistance gene for selection of transformed cells. CHO-k1 cells were transfected by CaPO₄ precipitation (Maniatis et al., 1982) or with the LipofectAMINE liposome reagent (Gibco/BRL). Clonal cells were obtained by dilution and screened by ligand binding and adenylyl cyclase activation (Granneman et al., 1993). Cell lines used in the study had similar levels of receptor expression (1-2 pmol/mg of protein) and adenylyl cyclase activation.

Radioligand binding. Ligand binding was performed with ¹²⁵I-CYP (DuPont NEN, Boston, MA) as described previously (Emorine et al., 1989). Briefly, culture medium was removed and cells were washed in phosphate-buffered saline, then harvested in 25 mM HEPES, pH 8.0, buffer containing 2 mM MgCl₂ and 1 mM EDTA. Cells were lysed and centrifuged at 48,000 × g for 15 min to obtain crude membranes. Membrane pellets were resuspended by homogenization and used directly, or frozen at 80° until used. Freezing did not affect binding. Membranes were resuspended in 75 mM Tris, pH 7.4, 12.5 mM MgCl₂, 2 mM EDTA, and 1 mM ascorbic acid. Saturation analysis was performed with concentrations of ¹²⁵I-CYP ranging from 65 pM to 4 nM, with 1 mM ISO used to define nonspecific binding. For competition studies, 100 µM desmethylimipramine was included in the incubation to reduce nonspecific binding (Emorine et al., 1989). Incubations were carried out in volume of 150 µl for 1 hr at 30°, and were terminated by vacuum filtration over glass fiber filters. K_i values were calculated from IC₅₀ values that were determined by nonlinear regression analysis of three to six experiments, each performed in triplicate.

cAMP accumulation assay. cAMP accumulation was performed in duplicate as previously described (Chaudhry et al., 1994). Briefly, cells grown in 24-well plates were washed two times in Ham's F-12 medium containing 1 mM IBMX and 0.1 mM ascorbic acid. After a 15-min preincubation in the above medium, cells were challenged with various agonists. Reactions were terminated after 30 min by the addition of perchloric or trichloroacetic acid. After neutralization, accumulated cAMP was determined by radioimmunoassay (Fransen and Krishna, 1976) or protein binding assay (Brown et al., 1971).

Adenylyl cyclase assay. Membrane adenylyl cyclase assays were performed by a modification (Granneman et al., 1991) of the method of Salomon et al. (1974). Briefly, membranes (5-15 µg of protein) were preincubated at 4° in a volume of 40 µl with the specified drugs for 15 min. Adenylyl cyclase reactions were initiated by addition of 10 µl of substrate mix and terminated after 30 min at 30°.

Data analysis. Ligand binding and adenylyl cyclase data were analyzed with Enzfitter software (Enzfitter; Elsevier Biosoft, Cambridge, UK). EC₅₀ values in cAMP accumulation assays were determined graphically. Antagonist binding affinity (K_B) was determined according to the equation K_B = [B]/[DR 1] where [B] is the antagonist concentration and the dose-ratio (DR) is the EC₅₀ value of the agonist in the presence of antagonist divided by the control EC₅₀ value. Values reported are mean ± standard error. Planned comparisons between means were evaluated with Bonferroni t test, with critical values of p < 0.05 (two-tailed) judged significant.

	Results

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The characteristic pharmacological properties of rat ₁- and ₃-AR that were evaluated are illustrated in Fig. 1. ISO, used as the reference catecholamine agonist, had a 100-fold higher affinity for ₁- versus ₃-AR in binding assays. Despite differences in binding afinity, ISO exhibited nearly similar potency in the stimulation of cAMP accumulation. Comparison of the binding and cAMP accumulation curves indicates that ISO fully activated adenylyl cyclase at concentrations that did not fully saturate the receptors. Although a discrepancy between K_i and EC₅₀ values would be expected in cells expressing high levels of receptors (Wilson et al., 1996; Leby et al., 1993), the difference was at least 50 times greater for the ₃-AR versus the ₁-AR in cells expressing similar levels of receptors (3.4 log units versus 1.7, respectively). The high efficiency of ₃-AR coupling to cAMP generation as indicated by large discrepancy between K_i and EC₅₀ occurs at various levels of receptor expression and is present in cells that natively express the receptor (Granneman, 1995; Wilson et al., 1996). Thus, like the ₂-AR (Leby et al., 1993), the ₃-AR has a high degree of coupling efficiency that is not matched by the ₁-AR (see also Green and Liggett, 1994).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Pharmacological properties of rat 1- and 3-AR expressed in CHO cells. Top, Interaction of ISO with 1- and 3-AR in binding and cAMP accumulation assays. Competition assays were performed in the presence of 100 pM (1-AR) or 500 pM (3-AR) 125I-CYP. Bottom, Activation of cAMP accumulation by CL and BRL. Values are means ± standard error (3 experiments) and are given as a percentage of the maximal response elicited by ISO.

₃-AR have received attention as a therapeutic target for antiobesity agents and certain phenethanolamines have been synthesized that selectively bind and activate this receptor. CL and BRL are prototypic phenethanolamine agonists that bound the rat ₃-AR with moderate affinity and activated the receptor with very high potency (Fig. 1, bottom). In contrast, BRL had moderate affinity for the ₁-AR but exhibited little agonist activity. CL did not bind or activate the ₁-AR at the concentrations employed.

The -AR subtypes and chimeric receptors were characterized with various adrenergic agonists in ligand binding and cAMP accumulation assays, and the results are summarized below and in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Pharmacological properties of native and chimeric rat 1- and 3-AR expressed in CHO cells

Agonist binding affinity. Exchange of the amino acids beyond TM6 had no effect on the binding of catecholamines. Thus, the ₁/₃ O3 chimera retained high affinity for NE, ISO, and DO characteristic of 1-AR, whereas the ₃/₁-O3 chimera retained low affinity characteristic of ₃-AR. In sharp contrast, substitution of sequence beyond TM6 (importantly TM7) had dramatic and reciprocal effects on the affinity of ₁- and ₃-AR for the prototypic ₃-AR agonists BRL and CL. The ₁/₃-O3 chimera exhibited a much higher affinity for BRL (20-fold) and CL (>80-fold) compared with native ₁-AR. The complementary chimera that substituted ₁-AR sequence in the ₃-AR had a 20-fold reduced affinity for BRL and a 125-fold lower affinity for CL.

cAMP accumulation. Catecholamines were equally potent in activating native ₁ AR and the 1/₃-O3 chimera. In sharp contrast, this substitution profoundly affected the potency of ₃-AR-selective ligands. Compared with ₁-AR, the ₁/₃-O3 chimera was 100 times more sensitive to the agonist actions of BRL, whereas the instrinsic activity relative to ISO was increased from 0.2 to 0.64 ± 0.11. The effects of the ₃-O3 substitution on CL action were more dramatic, with agonist potency being increased by more than 2500 times. Indeed, CL was essentially inactive at the ₁-AR, but was nearly as potent and efficacious (intrinsic activity = 0.96 ± 0.28) as norepinephrine in activating the ₁/₃-O3 chimera.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Alignment of the seventh TM segment of the rat 1- and 3-AR.

View this table: [in this window] [in a new window]   TABLE 2 Pharmacological properties of 1-F350A,F351L mutant receptors

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Adenylyl cyclase activation of CHO cell membranes containing rat 1-AR (top), 3-AR (bottom), and 1-F350A,F351L (middle). Concentration-response curves to ISO and CL were generated in the absence () and presence () of 100 nM ()-propranolol. Values are mean ± standard error (3 experiments).

	Discussion

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₁- and ₃-AR have distinct pharmacological properties, yet relatively little molecular genetic analysis has been performed that examines the binding specificity of ₃-AR. Guan et al. (1995) investigated the binding affinity of BRL in a series of chimera composed of human ₂- and ₃-AR. However, selectivity of BRL for ₃-AR was modest (about 10-fold), and effects on agonist activity were not addressed. In the present work, a series of chimeric and mutated ₁- and ₃-AR subtypes was constructed to identify regions of these molecules that confer subtype-specific signaling properties. This work took advantage of the greater differences between ₁- and ₃-AR in affinity for BRL, as well as the greater selectivity of CL, which has not been previously examined.

The major finding of this analysis is that the binding of and activation by prototypic phenethanolamine ₃-AR ligands was dramatically affected by alterations in TM7. Specifically, replacing ₁-AR TM7 with sequence derived from the ₃-AR conferred the ability of ₃-AR-selective ligands to bind and activate the receptor. Conversely, replacing ₃-AR TM7 with sequence derived from the ₁-AR dramatically reduced the affinity and potency of ₃-AR-selective agonists. It is important to note that these substitutions did not alter binding or activity of catecholamine agonists.

The effects of TM7 substitutions on ₃-selective agonist potency (EC₅₀) were far greater than could be accounted for by changes in binding affinity, indicating that this region is also critical for receptor activation by these ligands. In general, the effects of TM7 substitutions were greatest for CL, which exhibits the highest degree of selectivity for the ₃-AR. The improvement in CL action in the ₁/₃-O3 chimera is difficult to quantify because the compound was essentially inactive at the ₁-AR. Nevertheless, CL potency increased by more than 2500-fold in this chimera. The differential effects of TM7 substitution on affinity and potency are perhaps best illustrated in the ₃/₁-O3 chimera, in which CL potency was reduced 100 times more than binding affinity. Thus, although CL and ISO bound this chimera with equal affinity, the ability of CL to activate the receptor was two orders of magnitude less than ISO.

Site-directed mutagenesis demonstrated that substitution of Phe350 and Phe351 in TM7 to alanine and leucine, respectively, was sufficient to produce the phenotype seen with complete TM7 and carboxyl tail substitution. Like the ₁/₃-O3 chimera, the actions of catecholamines at the ₁-F350A,F351L mutant were indistinguishable from the native ₁-AR. Similarly, the mutation did not affect the ability of ()-propranolol to antagonize catecholamine or ₃-AR agonists. Nonetheless, mutation of Phe350 and Phe351 of the ₁-AR to residues found in the ₃-AR was sufficient to improve the affinity and potency of CL by more than 100- and 1000-fold, respectively.

How might mutation of Phe350 and Phe351 permit binding and activation by ₃-AR ligands without affecting the actions of catecholamines or propranolol? Binding depends upon direct interaction of the ligand with specific amino acids, as well as interactions among amino acids that form and stabilize the ligand binding pocket, but do not directly contact the ligand. Catecholamines are relatively small molecules whose binding is thought to be stabilized by specific interactions with amino acid side chains in TM3, TM4, and TM5 that are conserved in each of the -AR subtypes (Strader et al., 1987, 1988; Dixon et al., 1988; Strosberg et al., 1993; Blin et al., 1993). In contrast, ligands that selectively activate ₃-AR contain bulky alkylamine chains that presumably permit subtype-selective interactions. Strosberg et al. (1993) have used molecular modeling to suggest that extended conformations of selective ₃-AR ligands mediate agonist properties by contacting residues within the TM regions, including potential interactions with TM7. Based upon models of related receptors, Phe350 and Phe351 are predicted to be present near the beginning of TM7 in the ₁-AR and Phe351 is likely to face the ligand binding core of the receptor (Mizobe et al., 1996, Baldwin et al., 1997). It is therefore possible that Phe350 and Phe351 prevent activation of the ₁-AR by denying extended conformations of ₃-AR-selective ligands access to the ligand binding groove. Alternatively, the interaction of Phe350 and Phe351 with residues in other TM regions might alter the binding pocket available to the phenethanolamine agonists and thereby restrict activation indirectly (Mizobe et al., 1996). Such an affect on the binding pocket, however, would seem to be subtle, because the actions of catecholamines and propranolol were not affected in the F350A,F351L mutant. It is also conceivable that ₃-AR agonists interact directly with alanine and/or leucine in TM7, although additional interactions must be required because the ₂-AR has a similar sequence to the ₃-AR in this region (leucine in both positions), yet is not activated by CL (Bloom et al., 1992). In any event, the present results provide experimental support for a model in which the selectivity of prototypic ₃-AR ligands is conferred by the ability of these compounds to access a binding pocket formed with TM7.

₁-and ₃-AR can be distinguished by differences in coupling efficiency to cAMP generation (Leby et al., 1993; Green and Liggett, 1994; Granneman, 1995; Wilson et al., 1996). The high degree of coupling efficiency of ₃-AR requires intact cells, occurs over a large range of receptor expression levels and is independent of cell background (Granneman, 1995; Wilson et al., 1996). As generation of cAMP requires the interaction of multiple proteins in the context of an intact cell, modest changes in agonist potency can be difficult to interpret. Previous work by Green and Liggett (1994) attributed the low degree of coupling efficiency of ₁-AR to a proline-rich sequence in the third intracellular (I3) loop. Consistent with these results, replacement of TM5 through TM6 sequence with ₃-AR sequence, which contains I3, seemed to improve coupling efficiency of the ₁-AR. Indeed, the impact of TM5-6 replacement on coupling observed the present experiment (10-20-fold) was somewhat greater than that observed by Green and Liggett for the replacement of the proline-rich sequence alone, suggesting that differences in coupling efficiency involve the proline-rich region as well as additional sequences in I3 and perhaps TM5 and 6. Nevertheless, ₁-AR TM5-6 replacement failed to consistently suppress the coupling efficiency of the ₃-AR, indicating that this region alone is insufficient to account for subtype differences in coupling efficiency.

Several amino acids in the ₂-AR have been identified that are crucial for high affinity binding of and activation by catecholamines, including Asp113, Ser204, Ser207, Phe290, and Tyr326 (Strader et al., 1987, 1988; Dixon et al., 1988). These amino acids are conserved in the ₁- and ₃-AR and thus other determinants must be important in explaining the 30-50-fold difference in catecholamine binding affinity between these subtypes. However, the present study did not clearly identify these regions. Although replacement in ₁-AR of the sequence from TM5-6 with the ₃-AR sequence reduced catecholamine binding affinity by about 5-fold, including the homologous region of the ₁-AR did not increase affinity in the ₃-AR. Given that the catecholamine binding pocket is thought to be formed by the juxtaposing of TM regions, it is likely that several amino acids in the TM regions indirectly influence affinity by affecting the alignment or stabilization of the residues that interact directly with catecholamines. Thus, although ₃-AR substitutions disturb catecholamine binding in the context of the ₁-AR, the converse does not seem to be true.

In summary, the present work demonstrates that residues in TM7 are critical in conferring subtype-specific activation by ₃-AR-selective phenethanolamines. The ability of these residues to dramatically influence activation by prototypic ₃-AR-selective agonists without affecting catecholamine or propranolol action demonstrates that the sites critical for these interactions are distinct.