Introduction

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The _1b-adrenergic receptor (AR) belongs to the superfamily of G protein-coupled receptors (GPCR), which transduce signals across the cell membrane. Stimulation of the _1b-AR by catecholamines can activate proteins of the Gq/11 family, resulting in phospholipase C-mediated production of inositol phosphates (IP).

All GPCR sequences share the presence of seven transmembrane (TM) -helices connected by alternating intracellular and extracellular hydrophilic loops (Wess, 1997). The seven TM helices contribute to the formation of the ligand binding pocket, whereas amino acid sequences of the intracellular loops mediate the interaction of the receptor with a number of signaling and regulatory proteins, including G proteins, arrestins, and GPCR kinases.

Within the large superfamily of GPCRs, a small number of amino acids are highly conserved throughout evolution. The Glu/Asp-Arg-Tyr motif (E/DRY motif) located at the cytosolic end of helix 3 occurs in the majority of GPCRs belonging to the "rhodopsin-like" subfamily. The high degree of conservation of this motif suggests that it must play an important role in receptor function. Indeed, several experimental and modeling studies highlighted that both negatively and positively charged residues of the E/DRY are involved in the activation process of GPCRs (Oliveira et al., 1994; Wess, 1997).

Recent studies have demonstrated that mutations of the aspartate of the E/DRY motif can induce variable levels of constitutive (agonist-independent) activity for rhodopsin (Cohen et al., 1993; Acharya and Karnik, 1996), the _1b-AR (Scheer et al., 1996, Scheer et al., 1997), and the V2 receptor for vasopressin (Morin et al., 1998). Interestingly, mutations of the homologous aspartate in the gonadotropin-releasing hormone receptor into asparagine enhanced the agonist-induced receptor response (Arora et al., 1997). In the muscarinic M1 receptor, some mutations of the homologous aspartate dramatically decreased receptor expression (Lu et al., 1997). However, for those receptor mutants that were expressed, the agonist-induced receptor response was similar to if not greater than that of the wild-type receptor.

Site-directed mutagenesis studies that have targeted the conserved arginine of E/DRY in different GPCRs have so far mainly identified receptor mutants modestly or severely impaired in their ability to mediate a signaling response. Indeed, it has been demonstrated that mutations of this conserved arginine impaired the response mediated by GPCRs linked to different signaling pathways, including rhodopsin (Franke et al., 1992; Acharya and Karnik, 1996), the M1 and M2 cholinergic receptors (Zhu et al., 1994; Jones et al., 1995), the V2 vasopressin receptor (Rosenthal et al., 1993), the _1b-AR (Scheer et al., 1996), and the gonadotropin-releasing hormone receptor (GnRH-R) (Arora et al., 1997; Ballestreros et al., 1998). These findings suggest that the invariant arginine plays a pivotal role in receptor-mediated activation of downstream signaling proteins. However, the mechanistic role played by the arginine of the E/DRY sequence in receptor activation remains unknown.

Recently, we combined experimental and computer-simulated mutagenesis of the _1b-AR to build a theoretical model of receptor activation (Scheer et al., 1996; Scheer et al., 1997). The comparative molecular dynamics (MD) analysis of the wild-type _1b-AR and several constitutively active receptor mutants highlighted a network of hydrogen-bonding interactions among conserved polar residues forming a "polar pocket" near the cytosol (N63 in helix 1, D91 in helix 2, N344 and Y348 in helix 7) and R143 of the E/DRY motif. We suggested that 1) this set of interactions constrains the receptor in its ground state by controlling the degree of cytosolic exposure attainable by R143 and 2) the main role of R143 is to mediate receptor activation, allowing amino acids of the intracellular loops to attain the right configuration for the formation of a site with docking complementarity with the G protein.

In this study, we performed a series of conservative and nonconservative mutations of R143 to further elucidate its role in the activation process of the _1b-AR. Various substitutions of R143 resulted in receptor mutants with either increased constitutive activity, impairment, or complete loss of receptor-mediated response. Interestingly, all the mutants displayed increased affinity for agonist binding compared with the wild-type _1b-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants conducted using the allosteric ternary complex model suggests that mutations of R143 can drive the _1b-AR into different states, supporting the crucial role of this residue in the activation process of the receptor.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion Appendix References

COS-7 Cell Culture and Transfections. COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and gentamicin (100 µg/ml) and transfected using the DEAE-dextran method. The cDNA encoding the hamster _1b-AR (Cotecchia et al., 1992) and its mutants were subcloned in pRK5. For inositol phosphate determination, COS-7 cells (0.15 × 10⁶) were plated in 12-well plates. For phosphorylation assays, cells were plated in 100-mm dishes (3 × 10⁶ cells). The transfected DNA encoding the receptors was 0.5 to 3 µg/10⁶ cells.

Ligand Binding. Membrane preparations derived from cells expressing the _1b-AR or its mutants and ligand binding assays using [¹²⁵I]iodo-2-[-(4-hydroxyphenyl)-ethylaminomethyl]tetralone (HEAT) were performed as described previously (Cotecchia et al., 1992). Prazosin (10⁶ M) was used to determine nonspecific binding. [¹²⁵I]HEAT concentration was 250 pM for measuring receptor expression at a single concentration and 80 pM for competition binding analysis. Saturation analysis and competition curves were analyzed using Prism 2.0 (GraphPAD Software, San Diego, CA).

IP Measurement. Transfected cells were labeled for 12 h with myo-[³H]inositol at 4 µCi/ml in inositol-free DMEM supplemented with 1% fetal bovine serum. Cells were then preincubated for 10 min in PBS containing 20 mM LiCl, and then stimulated for 45 min with epinephrine. Total inositol phosphates were extracted and separated as described previously (Cotecchia et al., 1992).

³²P-Labeling and Immunoprecipitation of the Receptors. Transfected COS-7 cells were equilibrated in phosphate-free DMEM for 2 h and then incubated in the same buffer containing ³²P_i (0.2 mCi/ml) for 2 h at 37°C. The incubation was then continued in the absence or presence of epinephrine as indicated and immunoprecipitation of the phosphorylated receptors was performed as described previously (Lattion et al., 1994). After autoradiography, the ³²P content of the gel slices containing the immunoprecipitated receptor was quantified (cpm) by liquid scintillation counting. A separate set of dishes were incubated under similar conditions, but in the absence of ³²P_i to measure receptor binding. The receptors were expressed at similar levels and the ³²P content (cpm) of different gel slices was directly compared for statistical analysis.

Building of the Receptor Models and MD Simulations. Modeling of the 1b-AR was achieved following an ab initio procedure as described recently (Fanelli et al., 1998). The building of the receptor model consisted of an iterative procedure starting with a comparative MD study on the transmembrane domains of seven GPCRs (Fanelli et al., 1995) The starting arrangement of the seven helix bundle was successively modified and the model was complicated progressively by adding the intracellular and extracellular domains. A many-step iterative procedure, characterized by the experimental validation of the model in each of the upgrading steps, was employed. The most stringent validation of the model was achieved by challenging its capability to interpret and predict the functional properties of an ever-increasing number of 1b-AR mutants. The latest version of the model used in this study includes new experimental information derived from the electron micrographs of 3-D frog rhodopsin crystals (Unger et al., 1997). The tilt of the helices in the 1b-AR input arrangement previously obtained was slightly modified according to the tilt angles of the seven helices estimated from the map of frog rhodopsin (Baldwin et al., 1997; Unger et al., 1997). In addition, the third intracellular loop (i3) of the 1b-AR was completed by the addition of residues 236 to 284 (Fanelli et al., 1999b). Different input arrangements were built performing translations and rotations of the helices and loops, as well as modifications of side-chain torsion angles. Among the large number of arrangements tested, we selected the input structure of the 1b-AR that, upon MD simulations, produced an average arrangement showing the best agreement with the experimental data available on GPCRs together with high-quality check scores. The selected input structure of the wild-type 1b-AR was used to produce the input structures for the mutants using the molecular graphics package QUANTA 96 (Molecular Simulations, Waltham, MA).

Statistics. Results are expressed as mean ± S.E. Statistical significance was assessed by paired Student's t test.

Materials. COS-7 cells were from American Type Culture Collection (Rockville, MD); DMEM, gentamicin, fetal bovine serum and restriction enzymes from Life Technologies, Inc. (Grand Island, NY); Taq polymerase from Boehringer Mannheim (Mannheim, Germany); ¹²⁵I]HEAT and [³H]inositol from DuPont-New England Nuclear (Boston, MA); epinephrine, norepinephrine, phenylephrine, methoxamine, oxymetazoline, and dopamine were from Sigma (St. Louis, MO); and cirazoline and prazosin from Research Biochemical International (Natick, MA).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion Appendix References

Mutation of R143 into Alanine Counteracts the Effect of Constitutively Activating Mutations. We previously reported that the mutation of R143 to alanine profoundly impaired the _1b-AR-mediated IP response (Scheer et al., 1996). To further challenge the role of R143 in receptor activation, we introduced the mutation of R143 to alanine into either the D142A or the A293E constitutively active receptor. The constitutive activity of the receptor double mutants D142A/R143A and A293E/R143A was dramatically reduced compared with that of the D142A and A293E receptors. In addition, the epinephrine-induced IP response mediated by the D142A/R143A and A293E/R143A mutants was profoundly reduced compared with that of the wild-type _1b-AR (Table 1). Thus, the effect induced by the inactivating mutation of R143 into alanine was dominant over that induced by activating mutations of either D142 or A293, resulting in significant impairment of both the constitutive and agonist-induced activity of the receptor.

Effects of Mutations of R143 on the Receptor-Mediated IP Response. To further investigate its mechanistic role in receptor function, R143 was mutated into several amino acids differing in their charge, size, or hydropathy index (Table 1). All the mutated receptors were expressed in COS-7 cells at levels between 0.2 and 0.6 pmol/mg of protein. The receptor mutants were tested for their ability to mediate epinephrine-stimulated IP accumulation in transfected COS-7 cells.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   IP response of the 1b-AR and its mutants carrying substitutions of R143. COS-7 cells were transfected with the cDNA encoding the wild-type 1b-AR (WT) or its mutants R143K, R143H, R143D, R143A, R143N, R143I, and R143E. Cells were incubated in the absence (Bas) or presence of 104 M epinephrine (Epi) for 45 min. Total IP were measured as described under Experimental Procedures. Receptor expression measured in membrane preparations was in the range of 0.2 to 0.3 pmol/mg of protein for all receptors. The results are the mean ± S.E. of three independent experiments.

Phosphorylation Properties of Receptors Mutated at Position 143. A recent study reported that moderately uncoupled rhodopsin mutants carrying mutations of the arginine of the E/DRY motif displayed a higher level of light-induced phosphorylation than that of the wild-type receptor (Shi et al., 1998). Two of these mutants also displayed enhanced interactions with rhodopsin kinase and arrestin in the absence of 11-cis-retinal. These findings suggested the intriguing hypothesis that the lower level of transducin activation might result from enhanced phosphorylation and desensitization of the rhodopsin mutants. A small increase in basal phosphorylation was also reported for a V2 vasopressin receptor mutant, in which the mutation of the homologous arginine to histidine resulted in receptor uncoupling (Innamorati et al., 1997). Thus, to further characterize the properties of the _1b-AR mutants carrying mutations of R143, we measured the phosphorylation of the R143A, R143E, and R143K receptors in intact cells.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Phosphorylation of the 1b-AR and its mutants carrying substitutions of R143. COS-7 cells were transfected with the cDNA encoding the wild-type 1b-AR (WT) or its mutants R143A, R143E, and R143K. After labeling with 32Pi, cells were incubated in the absence (Bas) or presence of 104 M epinephrine (Epi) for 15 min. Immunoprecipitation of the phosphorylated receptors and quantification of their 32P content was performed as described under Experimental Procedures. Receptor expression was about 0.3 pmol/mg of proteins for all receptors. Receptor phosphorylation is expressed as percent of the control, which indicates the 32P content (cpm) of the WT receptor in the absence of epinephrine. The results are the mean ± S.E. of four independent experiments. a, P < .05 compared with the basal phosphorylation of each respective receptor; b, P < .05 compared with the basal phosphorylation of the WT.

Structural-Dynamic Features of the Receptors Carrying Mutations of R143. To further elucidate the structural and dynamic role of R143, MD simulations were performed on the _1b-AR model carrying mutations at R143. We have used a recently described upgraded three-dimensional model of the _1b-AR that incorporates several new pieces of experimental information available on GPCRs (Fanelli et al., 1998, 1999b). To gain further insight into the receptor-G protein interface, the up-graded _1b-AR model included the entire i3 loop of the receptor (residues 236-284).

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Receptor models. The four panels display the results of MD simulations carried on the wild-type 1b-AR (ground state), the constitutively active mutants A293E and R143K, and the activation-deficient mutant R143E. All views are in a direction perpendicular to the helix main axis. The seven helices 1, 2, 3, 4, 5, 6, and 7 are colored in blue, orange, green, pink, yellow, sky blue, and violet, respectively. The i1, i2, and i3 loops are colored in green, white, and cyclamen, respectively, whereas the e1, e2, and e3 loops are colored in carnation. Each panel shows two views of the average minimized structure of each receptor together with several amino acids (colored according to their location) in the environment of the residue at position 143. In the middle of each panel, the solvent-accessible surface areas computed on i2, i3, and the cytosolic extensions of helices 5 and 6 are displayed.

Effects of Mutations at Position R143 on the Ligand Binding Properties of the Receptor. To further characterize the receptor mutants at R143, ligand binding studies were performed on membranes from COS-7 cells expressing the wild-type _1b-AR or the mutated receptors (Table 1). Saturation binding analysis of [¹²⁵I]HEAT indicated that the K_d values were similar at the wild-type and mutated receptors, ranging from 80 to 120 pM (results of two independent experiments not shown). In contrast, the affinity of various agonists differed among the receptors.

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Comparison of the Constitutively Active Receptors R143K and A293E with the Inactive Mutant R143E. To better understand the nature of the increased affinity for agonist binding of the activation-defective R143 mutants, we compared the ligand-binding properties of the inactive receptor R143E with those of the two constitutively active mutants, R143K and A293E. First, we assessed the intrinsic activities of seven structurally different agonists; second, we measured the binding affinity of the various agonists at the wild-type _1b-AR, R143E, R143K, and A293E receptors.

View this table: [in this window] [in a new window]   TABLE 2 IP accumulation was measured in Rat-1 cells permanently expressing the wild-type 1b-AR at 1 pmol/mg of protein after stimulation with agonists at a concentration of 104 M (epinephrine, norepinephrine, cirazoline, and oxymetazoline) or 103 M (phenylephrine, methoxamine, and dopamine) for 45 min. The intrinsic activity (IA) of the agonists was computed by dividing the maximal IP response induced by each ligand with that of epinephrine. The IA values are from three independent experiments. The Ki of different agonists was assessed in membranes from COS-7 cells expressing the wild type 1b-AR or its mutants A293E, R143K, and R143E at about 0.2 pmol/mg of protein. The Ki values are from two to five independent experiments that did not differ by more than 40%.

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View larger version (23K): [in this window] [in a new window]   Fig. 4.   Relationship between the intrinsic activity and the affinity shift for various ligands at the receptor mutants A293E, R143K, and R143E. The values of Ki and intrinsic activities are from Table 2. The affinity shift, expressed as Ln units, is the ratio between the Ki value at the mutated and wild-type 1b-ARs. The experimental results were overlaid on the theoretical curves shown in Fig. 5 in the Appendix.

Analysis of the Relation Between Ligand Intrinsic Activity and Shift of Affinity. As explained in the Appendix, the two-state allosteric model of receptor activation predicts that if a mutation alters the stability constant for the conversion of the inactive (R) to the active state (R*) (and thus basal activity), it also changes ligand binding affinity. The extent and direction of such an "allosteric" shift in affinity depends on ligand efficacy; therefore, it must be related to the intrinsic activity of the ligand (Samama et al., 1993).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion Appendix References

In this study, we combined site-directed mutagenesis of the _1b-AR, computational simulations of receptor dynamics, and thermodynamic analysis of the receptor's pharmacological properties to provide hypotheses about the role played by R143 belonging to the conserved E/DRY motif in the activation process of the receptor.

Role of R143 in Receptor Activation. Several lines of evidence support the conclusion that R143 of the _1b-AR is an important residue in the process of receptor activation. First, the inactivating mutation of R143 to alanine severely impaired both the constitutive and agonist-induced activity of the A293E and D142A mutants (Table 1). Secondly, a number of mutations of R143 resulted in a profound impairment of the agonist-induced receptor response (Fig. 1). Finally, the fact that the R143A and R143E mutants were unable to undergo agonist-induced phosphorylation seems to exclude enhanced phosphorylation and desensitization as the main mechanism underlying the decrease in the receptor-mediated response for the mutants carrying neutral or negative amino acids at position 143.

R143 Plays a Role in the Isomerization of the Receptor. Our findings provide evidence that mutations of R143 enhance the stability constant (J), which governs the transition of the _1b-AR between the inactive (R) and active (R*) states (Samama et al., 1993).

View larger version (36K): [in this window] [in a new window]   Fig. 5.   A, relationship between apparent intrinsic activity and "true" thermodynamic efficacy according to the two-state allosteric model. Data were generated according to eqs. 2b. Maximal effects for ligands of different efficacy (, as given on the x-axis) were computed for different values of J (shown as log values in the legend) and converted into "relative" intrinsic activity by dividing all of them for the Emax of the ligand with the highest efficacy ( =1000). B, effect of changing J on the relation between shift in affinity and relative intrinsic activity. Shifts were calculated (eq. 4b) for different values of J (legend) and are relative to that of "wild-type" receptor (J=0.0001) for ligands of different efficacy as in A. Relative intrinsic activity (calculated as in A) refers to the "wild-type" receptor.

Conclusions. Our findings demonstrate that different mutations of R143 in the _1b-AR result in apparently divergent functional effects. However, the results of thermodynamic analysis and of MD simulations of the receptor mutants could provide some hypothesis suggesting that R143 in the _1b-AR plays a role in receptor isomerization toward different states, which are best represented by the mutants R143K and R143E. The mutation of R143 to lysine seems to trigger receptor isomerization to an "active" state characterized by high affinity for agonists, increased basal and agonist-induced activity, enhanced basal and agonist-stimulated phosphorylation, and receptor conformers that predict productive receptor-G protein coupling. On the contrary, the mutation of R143 to glutamic acid seems to induce the isomerization of the receptor to an "active-like" state that shares some properties with the "active" forms (including high affinity for agonists, enhanced basal phosphorylation, and the opening of a cytosolic crevice between the i2 and i3 loops) but is severely impaired in its ability to mediate a response. The other mutants of R143 share features of both the R143K and R143E receptors.