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• W1986184460 abstract "To investigate their role in receptor coupling to Gq, we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the α1b-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the α1b-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg254 and Lys258 in the third intracellular loop and Lys291 at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val147 and Leu151 in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg254, Lys258, as well as Leu151 are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the α1b-AR. These findings contribute to the delineation of the molecular determinants of the α1b-AR/Gq interface. To investigate their role in receptor coupling to Gq, we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the α1b-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the α1b-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg254 and Lys258 in the third intracellular loop and Lys291 at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val147 and Leu151 in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg254, Lys258, as well as Leu151 are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the α1b-AR. These findings contribute to the delineation of the molecular determinants of the α1b-AR/Gq interface. adrenergic receptor(s) guanyl nucleotide binding regulatory protein G protein-coupled receptor inositol phosphate second and third intracellular loop Dulbecco's modified Eagle's medium [125I]iodo-2-[β-(4-hydroxyphenyl)ethylaminomethyl]tetralone molecular dynamics phospholipase C polymerase chain reaction root mean square deviation constitutively active mutant inactive and active states, respectively The α1b-adrenergic receptor (α1b-AR)1belongs to the superfamily of G protein-coupled receptors (GPCRs) that transmit a variety of signals across the cell membrane. Stimulation of the α1b-AR by catecholamines activates proteins of the Gq family, resulting in the production of inositol phosphates (IP) via the activation of phospholipase C (PLC) (1Wu D. Katz A. Lee C.H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar). GPCRs are structurally characterized by seven transmembrane α-helices connected by alternating extracellular (e) and intracellular (i) loops. While the extracellular portion of these receptors is primarily involved in ligand binding, the cytosolic loops mediate the interaction of the receptors with a number of signaling and regulatory proteins, including G proteins, arrestins, and G protein-coupled receptor kinases (reviewed in Ref. 2Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (511) Google Scholar). Evidence suggests that a conformational adjustment within the helical bundle of the receptor underlies the process of agonist-induced activation of GPCRs (reviewed in Ref. 3Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar). The current hypothesis is that the transition from the inactive (R) to active (R*) state of a GPCR results in receptor interaction with, and activation of, a G protein. Thus a GPCR-mediated biological response involves a series of events (i.e. receptor activation, receptor-G protein interaction, and receptor-induced G protein activation) for which a detailed mechanism still remains elusive at the molecular level. Although residues located in the helical bundle and at the boundary between the membrane and the cytosol may play a role in the “conformational switch” underlying receptor activation, amino acids in the intracellular loops are believed to be more directly involved in receptor-G protein interaction and/or receptor-induced G protein activation. The combination of these two latter events, which cannot be unequivocally separated experimentally, is generally indicated with the term of receptor-G protein coupling. We have previously provided evidence that the negatively and positively charged amino acids of the conserved DRY motif at the cytosolic end of helix 3 play a key role in the activation process of the α1b-AR (4Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (361) Google Scholar, 5Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar, 6Scheer A. Costa T. Fanelli F. De Benedetti P.G. Mhaouty-Kodja S. Abuin L. Nenniger-Tosato M. Cotecchia S. Mol. Pharmacol. 2000; 57: 219-231PubMed Google Scholar). Following a combination of experimental and computer-simulated mutagenesis of the α1b-AR, we have hypothesized that protonation of the aspartate (Asp142) and a shift of the arginine (Arg143) out of a conserved “polar pocket” are crucial steps in the transition of the receptor from the inactive (R) to active (R*) state (4Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (361) Google Scholar, 5Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar, 6Scheer A. Costa T. Fanelli F. De Benedetti P.G. Mhaouty-Kodja S. Abuin L. Nenniger-Tosato M. Cotecchia S. Mol. Pharmacol. 2000; 57: 219-231PubMed Google Scholar). Several studies have tried to identify the amino acids of different GPCRs involved in G protein coupling at both experimental (as reviewed in Ref. 2Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (511) Google Scholar) and theoretical levels (7Lichtarge O. Bourne H.R. Cohen F.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7507-7511Crossref PubMed Scopus (169) Google Scholar, 8Fanelli F. Menziani C. Scheer A. Cotecchia S. De Benedetti P.G. Proteins. 1999; 37: 145-156Crossref PubMed Scopus (37) Google Scholar, 9Oliveira L. Paiva A.C. Vriend G. Protein Eng. 1999; 12: 1087-1095Crossref PubMed Scopus (65) Google Scholar, 10Horn F. van Der Wenden E.M. Oliveira L., AP, I.J. Vriend G. Proteins. 2000; 41: 448-459Crossref PubMed Scopus (50) Google Scholar). The majority of these studies indicate that sequences in the i2 loop as well as in the N and C termini of the i3 loop play an important role in the efficiency of receptor-G protein coupling and/or in the selectivity of receptor-G protein recognition. BBXXB or BBXB motifs located in different cytosolic loops (where B is any basic amino acid and X is any residue) have been implicated in the coupling of a number of GPCRs to G proteins (11Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar, 12Hogger P. Shockley M.S. Lameh J. Sadee W. J. Biol. Chem. 1995; 270: 7405-7410Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 13Lee N.H. Geoghagen N.S. Cheng E. Cline R.T. Fraser C.M. Mol. Pharmacol. 1996; 50: 140-148PubMed Google Scholar). However, this motif has not been found to be universally important for all GPCRs. Other studies have identified hydrophobic amino acids as contributing to the receptor-G protein interface (14Moro O. Lameh J. Hogger P. Sadee W. J. Biol. Chem. 1993; 268: 22273-22276Abstract Full Text PDF PubMed Google Scholar, 15Arora K.K. Sakai A. Catt K.J. J. Biol. Chem. 1995; 270: 22820-22826Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 16Wade S.M. Scribner M.K. Dalman H.M. Taylor J.M. Neubig R.R. Mol. Pharmacol. 1996; 50: 351-358PubMed Google Scholar). In conclusion, what has become abundantly clear is that there is no simple sequence determinant that can be attributed to receptor-G protein coupling. In a recent modeling study (8Fanelli F. Menziani C. Scheer A. Cotecchia S. De Benedetti P.G. Proteins. 1999; 37: 145-156Crossref PubMed Scopus (37) Google Scholar), docking simulations between active forms of the α1b-AR and a Gq heterotrimer led us to suggest that the positive surface of the cytosolic portion of GPCRs could complement a negative surface found on different G protein α subunits and thereby play a role in receptor-G protein coupling. However, the docking simulations also suggested that, despite the large number of cationic amino acids, only some might interact with anionic residues in the Gq α-subunit. To investigate the role of cationic residues in receptor-G protein coupling, we have mutated all the basic amino acids located in the i1, i2, and i3 loops of the α1b-AR and investigated the effect of these mutations on receptor-mediated production of IP. In addition, we have also characterized the effects resulting from mutations of conserved hydrophobic residues in the cytosolic portion of the receptor. Our findings demonstrate that mutations of the basic residues in the cytosolic portion of the receptor have wide ranging phenotypes. Only mutations of three (Arg254, Lys258, and Lys291) out of the twenty-three basic amino acids studied impaired the receptor-mediated signaling response. We also demonstrate an important role for two highly conserved hydrophobic residues in receptor function. The effect of these mutations has been evaluated in the context of both the ab initio model previously described (17Fanelli F. Menziani C. Scheer A. Cotecchia S. De Benedetti P.G. Methods. 1998; 14: 302-317Crossref PubMed Scopus (42) Google Scholar) and a new model of the α1b-AR built on the recently solved 2.8-Å crystal structure of rhodopsin (18Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5048) Google Scholar). COS-7 cells were from the American Type Culture Collection (Rockville, MD); DMEM, gentamicin, fetal bovine serum, and restriction enzymes from Life Technologies, Inc. (Grand Island, NY);Pwo polymerase was from Roche Molecular Biochemicals(Mannheim, Germany); [125I]HEAT and [3H]inositol from PerkinElmer Life Sciences (Boston, MA); epinephrine was from Sigma Chemical Co. (St. Louis, MO), and prazosin from Research Biochemical International. The cDNA of the hamster α1b-AR (19Cotecchia S. Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1633-1639Abstract Full Text PDF PubMed Google Scholar) was mutated using PCR-mediated mutagenesis and Pwo DNA polymerase. The constructs were subcloned in the pRK5 expression vector, and mutations were confirmed by automated DNA sequencing (Microsynth GmbH, Switzerland). COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum and gentamicin (100 μg/ml) and transfected using the DEAE-dextran method. For inositol phosphate determination, COS-7 cells (0.15 × 106) were seeded in 12-well plates. The quantity of transfected receptor encoding DNA was 0.3–3 μg/106 cells. Membrane preparations derived from cells expressing the α1b-AR or its mutants and ligand binding assays using [125I]HEAT were performed as previously described (19Cotecchia S. Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1633-1639Abstract Full Text PDF PubMed Google Scholar). Prazosin (10−6m) was used to determine nonspecific binding. [125I]HEAT at a concentration of 250 pm was used for measuring receptor expression at a single concentration and 80 pm for competition binding analysis. Saturation analysis and competition curves were analyzed using Prism 3.02 (GraphPad Software Inc., San Diego, CA). 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 preincubated for 10 min in phosphate-buffered saline containing 20 mm LiCl and then stimulated for 45 min with different concentrations of epinephrine from 10−10 to 10−4m. Total inositol phosphates were extracted and separated as described previously (19Cotecchia S. Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1633-1639Abstract Full Text PDF PubMed Google Scholar). Ab initio modeling of the α1b-AR receptor was achieved following the iterative procedure previously described (17Fanelli F. Menziani C. Scheer A. Cotecchia S. De Benedetti P.G. Methods. 1998; 14: 302-317Crossref PubMed Scopus (42) Google Scholar). The wild type α1b-AR input structure was selected from among over 200 tested input arrangements according to both internal and external consistency criteria and was used to produce the input structures for the receptor mutants. These structures were obtained by substituting the mutated residue in the wild type input structure by means of the molecular graphics package QUANTA (release 98; Molecular Simulations Inc., Waltham, MA). Minimization and molecular dynamics (MD) simulations of the receptor models were performed using the program CHARMm (Molecular Simulations Inc), following the computational protocol previously described (17Fanelli F. Menziani C. Scheer A. Cotecchia S. De Benedetti P.G. Methods. 1998; 14: 302-317Crossref PubMed Scopus (42) Google Scholar). In a previous study MD runs of 1050 ps were performed to compare the dynamic features of the wild type receptor with those of the constitutively active mutants (8Fanelli F. Menziani C. Scheer A. Cotecchia S. De Benedetti P.G. Proteins. 1999; 37: 145-156Crossref PubMed Scopus (37) Google Scholar). Because the first 100 ps of the equilibrated trajectory were sufficiently representative of the whole trajectory and given the high number of mutants considered in this study, MD runs of 150 ps were generally performed following the same heating and equilibration set-up as that employed for the longer MD simulations. The results reported were collected every 0.5 ps during the last 100 ps of the equilibrated MD trajectory. Finally, for each mutant the structure averaged over the 200 structures stored during the production phase were used for the comparative analysis. The average minimized structure of the wild type α1b-AR showed a root mean square deviation (r.m.s.d.) of 3.94 Å from the rhodopsin structure, the deviation being larger (r.m.s.d. = 4.85 Å) before calculations. r.m.s.d. levels were computed by superimposing the main chain atoms of segments 37–62, 74–100, 111–133, 152–171, 202–225, 253–276, and 286–306, representing the seven transmembrane helices of rhodopsin, with those in the homologous segments 45–70, 82–107, 119–141, 161–180, 202–225, 295–318, and 328–348 of the average minimized structure of the wild type α1b-AR. Another model of the α1b-AR was built by comparative modeling (20Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10609) Google Scholar) using the recently determined 2.8-Å x-ray structure of rhodopsin (18Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5048) Google Scholar) as a template. Eight different chimeric α1b-AR/rhodopsin templates (shown in the supplementary material) were constructed in which the e2, the i3, and in some cases only the i2 loop were extracted from the input structure of theab initio model of the α1b-AR. Furthermore, in the chimeras helix 5 has been elongated by 10 amino acids using the α1b-AR sequence after deleting the 226–235 rhodopsin segment. Finally, an α-helical segment of 6 amino acids using the α1b-AR sequence has been added to the N terminus of the helix 6 of rhodopsin after deleting the 240–248 rhodopsin segment. For each of the eight different templates, MODELLER generated 25 models. Among the 200 models finally obtained, 20 models were selected showing low restraint violations and low numbers of main-chain and side-chain bad conformations or close contacts. These models were completed by the addition of the polar hydrogen's and subjected to automatic and manual rotation of the side-chain torsion angles when in bad conformations, as well as to energy minimization and MD simulations according to the computational protocol employed for simulating the ab initioα1b-AR model. Different combinations of intra-helix distance constraints were also probed. About 450 MD trial runs were done to select the proper input structure for the wild type α1b-AR. The final input structure selected that was obtained using the alignment (see the supplementary material) was then used for generating the input structures for the receptor mutants. The structures of the wild type receptor and its mutants averaged over the last 100 ps of the 150 ps MD trajectory were finally minimized and considered for the comparative analysis. The input structure of the wild type α1b-AR showed an r.m.s.d. of 0.17 Å from the rhodopsin structure (r.m.s.d. was computed by employing the matching criteria described above for the ab initio model). This deviation increases to 2.07 Å upon energy minimization and MD simulations, becoming quite close to the value that would be expected given a sequence identity of 22.4% between the transmembrane segments of the α1b-AR and rhodopsin (21Chothia C. Lesk A.M. EMBO J. 1986; 5: 823-826Crossref PubMed Scopus (1988) Google Scholar). The wild type and mutated α1b-ARs were expressed in COS-7 cells and tested for their ability to bind the radioligand [125I]HEAT and epinephrine. Saturation binding experiments indicated that theK D of [125I]HEAT was ∼80 pm for all the receptors studied (results not shown), whereas the IC50 values for epinephrine varied as indicated in Table I. The affinity of prazosin for the different receptor mutants was similar to that for the wild type α1b-AR (results not shown). Receptor coupling to the Gq/PLC pathway was assessed as the ability of the receptor mutants to mediate epinephrine-stimulated IP accumulation (Table I). Transfections using 3 μg of DNA per 1 × 106 cells resulted in the expression of all receptor mutants at levels ranging from 60 to over 250 fmol/well. In each experiment, the wild type α1b-AR was expressed using varying quantities of DNA (0.3, 1.3, and 3 μg of DNA/1 × 106 cells) resulting in low (between 60 and 100 fmol/well), medium (between 100 and 200 fmol/well), and high (between 200 and 300 fmol/well) levels of expression. This allowed us to always be able to directly compare the properties of the mutated receptors with those of the wild type α1b-AR expressed at comparable levels within the same experiment.Table IFunctional properties of the α 1b -AR and its mutantsReceptorExpressionBasal IPEpi-stimulated IPIC50 EpiEC50 Epifmol/well%%μmnmWT (high expression)264 ± 3636 ± 16365 ± 125.9 ± 0.3638 ± 6WT (ave. expression)158 ± 1033 ± 16303 ± 50WT (low expression)63 ± 821 ± 6148 ± 30D142A110 ± 27269 ± 891-ap < 0.05 paired Student's t test.634 ± 1360.22 ± 0.061-ap < 0.05 paired Student's t test.ND1-bND, not determined.A293E121 ± 73352 ± 431-ap < 0.05 paired Student's t test.852 ± 1041-ap < 0.05 paired Student's t test.0.1 ± 0.021-ap < 0.05 paired Student's t test.NDR74E182 ± 2210 ± 6296 ± 237.3 ± 0.238 ± 3H75E180 ± 3716 ± 7267 ± 215.9 ± 0.956 ± 2R77E87 ± 1529 ± 2248 ± 335.7 ± 0.3110 ± 25R74E/R77E28 ± 411 ± 884 ± 125.7 ± 0.514 ± 6R74E/H75E93 ± 1024 ± 7217 ± 834.9 ± 0.810 ± 6R148E198 ± 16219 ± 421-ap < 0.05 paired Student's t test.537 ± 721-ap < 0.05 paired Student's t test.2.6 ± 0.848 ± 15R159E232 ± 3938 ± 7635 ± 2101-ap < 0.05 paired Student's t test.2.7 ± 0.828 ± 10R160E285 ± 3682 ± 40528 ± 761-ap < 0.05 paired Student's t test.7.4 ± 0.728 ± 5R148E/R159E/R160E77 ± 21180 ± 961-ap < 0.05 paired Student's t test.636 ± 1701-ap < 0.05 paired Student's t test.7.4 ± 0.519 ± 2K161E79 ± 1661 ± 311037 ± 4751-ap < 0.05 paired Student's t test.3.1 ± 0.0633 ± 7K231E182 ± 41117 ± 241-ap < 0.05 paired Student's t test.678 ± 561-ap < 0.05 paired Student's t test.4.1 ± 0.316 ± 3R232E153 ± 3553 ± 3632 ± 701-ap < 0.05 paired Student's t test.2.4 ± 0.674 ± 20K235E121 ± 2720 ± 4360 ± 275 ± 0.223 ± 3K243E145 ± 5214 ± 9284 ± 836.4 ± 0.2100 ± 15K249E147 ± 4331 ± 9881 ± 551-ap < 0.05 paired Student's t test.3.9 ± 1.132 ± 10R254E187 ± 358 ± 5109 ± 161-ap < 0.05 paired Student's t test.2.9 ± 0.8NDR254A118 ± 388 ± 4125 ± 151-ap < 0.05 paired Student's t test.3.5 ± 0.121 ± 7K258E179 ± 3914 ± 5165 ± 797.3 ± 0.6NDK258A103 ± 3127 ± 20356 ± 835 ± 0.0348 ± 12R254E/K258E162 ± 4215 ± 530 ± 121-ap < 0.05 paired Student's t test.10 ± 0.3NDR254A/K258A125 ± 3711 ± 781 ± 201-ap < 0.05 paired Student's t test.4.4 ± 0.5NDR254E/K258E/D142A79 ± 314 ± 325 ± 51-ap < 0.05 paired Student's t test.0.22 ± 0.061-ap < 0.05 paired Student's t test.NDR254E/K258E/A293E177 ± 2716 ± 445 ± 121-ap < 0.05 paired Student's t test.0.12 ± 0.011-ap < 0.05 paired Student's t test.NDK269E127 ± 3932 ± 20387 ± 1326.4 ± 0.670 ± 8K271E70 ± 3013 ± 18344 ± 681-ap < 0.05 paired Student's t test.6.1 ± 0.522 ± 5R276E82 ± 2810 ± 7327 ± 521-ap < 0.05 paired Student's t test.7.2 ± 0.0388 ± 9K282E182 ± 3411 ± 8314 ± 1186.2 ± 0.0625 ± 7K285E116 ± 3219 ± 4493 ± 1411-ap < 0.05 paired Student's t test.3.3 ± 0.325 ± 5R288A130 ± 2847 ± 12280 ± 1843.8 ± 0.423 ± 6R288E134 ± 3055 ± 6266 ± 544.4 ± 0.0657 ± 4K290E113 ± 3453 ± 12565 ± 901-ap < 0.05 paired Student's t test.1.3 ± 0.234 ± 5K291A142 ± 2914 ± 4107 ± 171-ap < 0.05 paired Student's t test.8 ± 1.5NDK291E177 ± 444 ± 277 ± 241-ap < 0.05 paired Student's t test.6.3 ± 0.7NDR288A/K291A112 ± 3126 ± 9117 ± 71-ap < 0.05 paired Student's t test.3.1 ± 0.2NDR288E/K291E161 ± 314 ± 181 ± 191-ap < 0.05 paired Student's t test.1.7 ± 0.2NDR288E/K291E/D142A102 ± 4120 ± 676 ± 221-ap < 0.05 paired Student's t test.0.22 ± 0.031-ap < 0.05 paired Student's t test.NDR288E/K291E/A293E137 ± 2948 ± 10299 ± 760.45 ± 0.11-ap < 0.05 paired Student's t test.NDK294E118 ± 389 ± 5372 ± 845.3 ± 0.0357 ± 15Y144A62 ± 579 ± 201-ap < 0.05 paired Student's t test.767 ± 1491-ap < 0.05 paired Student's t test.3.5 ± 0.422 ± 8V147A94 ± 13440 ± 1601-ap < 0.05 paired Student's t test.744 ± 1731-ap < 0.05 paired Student's t test.0.03 ± 0.011-ap < 0.05 paired Student's t test.86 ± 15V147E66 ± 77 ± 38 ± 61-ap < 0.05 paired Student's t test.0.02 ± 0.011-ap < 0.05 paired Student's t test.NDV147E/A293E84 ± 188 ± 346 ± 81-ap < 0.05 paired Student's t test.0.06 ± 0.011-ap < 0.05 paired Student's t test.NDL151A169 ± 3328 ± 4114 ± 71-ap < 0.05 paired Student's t test.5.3 ± 0.1NDL151D208 ± 3416 ± 650 ± 171-ap < 0.05 paired Student's t test.6.9 ± 0.8NDL151D/A293E161 ± 1048 ± 12134 ± 121-ap < 0.05 paired Student's t test.0.04 ± 0.011-ap < 0.05 paired Student's t test.NDThe wild type α1b-AR (WT) and its mutants were expressed in COS-7 cells. Receptor expression was measured using 250 pm of [125I]HEAT on membrane preparations derived from transfected cells from one well of a six-well dish (approximately 150 μg of protein). Inositol phosphate (IP) accumulation was measured following incubation in the absence (Basal) or presence of 100 μm epinephrine (Epi-stimulated) for 45 min. The IP accumulation is expressed as the percentage increase in IP levels above those of mock transfected cells. Results for receptor expression and IP accumulation are the mean ± S.E. of at least three independent experiments. The IC50 for epinephrine was assessed in competition binding experiments using 80 pm of [125I]HEAT. The IC50 values are from thirty and three independent experiments for the wild type and mutated receptors, respectively. The EC50 values are from fifteen and two independent experiments for the wild type and mutated receptors, respectively.1-a p < 0.05 paired Student's t test.1-b ND, not determined. Open table in a new tab The wild type α1b-AR (WT) and its mutants were expressed in COS-7 cells. Receptor expression was measured using 250 pm of [125I]HEAT on membrane preparations derived from transfected cells from one well of a six-well dish (approximately 150 μg of protein). Inositol phosphate (IP) accumulation was measured following incubation in the absence (Basal) or presence of 100 μm epinephrine (Epi-stimulated) for 45 min. The IP accumulation is expressed as the percentage increase in IP levels above those of mock transfected cells. Results for receptor expression and IP accumulation are the mean ± S.E. of at least three independent experiments. The IC50 for epinephrine was assessed in competition binding experiments using 80 pm of [125I]HEAT. The IC50 values are from thirty and three independent experiments for the wild type and mutated receptors, respectively. The EC50 values are from fifteen and two independent experiments for the wild type and mutated receptors, respectively. Fig. 1 shows the localization of the amino acids mentioned in this study within a simplified topographical scheme of the α1b-AR based on its sequence alignment with bovine rhodopsin. The mutated amino acids are colored according to the functional effects induced upon their mutation. The first intracellular loop of the α1b-AR as with most GPCRs is short, being predicted to consist of just six amino acids (Fig. 1). Within this region there are three basic residues (Arg74, His75, and Arg77) forming a BBXXB/BBXB motif that has been described as important in the coupling of some receptors to G proteins. The individual mutations of Arg74, His75, and Arg77 into Glu did not result in any significant change in the ligand binding properties of the receptors or in their ability to mediate epinephrine-induced IP accumulation (TableI). To investigate whether the loss of more than a single basic residue had a greater effect than the single mutations, we generated the double mutants R74E/H75E and R74E/R77E. Both mutants displayed decreased levels of expression. However, their ability to mediate an agonist-induced IP response did not significantly differ from that of the wild type α1b-AR expressed at similar levels (TableI). It may therefore be concluded that the basic residues forming the BBXB motif in the i1 loop of the α1b-AR do not play a significant role in receptor-G protein coupling. These findings are in agreement with those from other studies on various GPCRs indicating that amino acids in the i1 loop are not important (22Shi W. Osawa S. Dickerson C.D. Weiss E.R. J. Biol. Chem. 1995; 270: 2112-2119Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 23Jin H. Nip S. O'Dowd B.F. George S.R. Biochim. Biophys. Acta. 1998; 1402: 165-170Crossref PubMed Scopus (4) Google Scholar) or only play a modest role (24Dixon R.A. Sigal I.S. Strader C.D. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 487-497Crossref PubMed Google Scholar, 25Mathi S.K. Chan Y. Li X. Wheeler M.B. Mol. Endocrinol. 1997; 11: 424-432Crossref PubMed Scopus (56) Google Scholar) in receptor-G protein coupling. The 13 amino acids that constitute the i2 loop of the α1b-AR and the cytosolic extension of helix 4 contain four cationic amino acids, Arg148, Arg159, Arg160, and Lys161. Within this region is found the DRYXX(V/I)XXXL motif identified as a common feature in the rhodopsin family of GPCRs and an essential part of the receptor activation mechanism (5Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar, 6Scheer A. Costa T. Fanelli F. De Benedetti P.G. Mhaouty-Kodja S. Abuin L. Nenniger-Tosato M. Cotecchia S. Mol. Pharmacol. 2000; 57: 219-231PubMed Google Scholar). Mutations of the four positively charged residues, Arg148, Arg159, Arg160, and Lys161, did not result in any change in the ligand binding properties of the receptor mutants (TableI). Only the triple mutant R148E/R159E/R160E displayed a 9-fold increase in affinity for epinephrine (Table I). Significantly, all the mutations resulted in an increased maximal epinephrine-stimulated activity of the receptor. However, the EC50 values of epinephrine for all the receptor mutants were similar to that of the wild type α1b-AR (Table I). The R148E mutant also displayed a significant 6-fold increase in its constitutive activity (Table I). Interestingly, when the sequence of the α1b-AR is aligned with those of the muscarinic M1, M3, and M5 receptors a homologous arginine is similarly located. Mutation of this arginine in the M5 muscarinic receptor to either Asp or Glu also produced constitutive activity (26Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1998; 273: 24322-24327Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The mutations of Ar" @default.
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