Human beta 2-adrenergic receptors expressed in Escherichia coli membranes retain their pharmacological properties

Proc. Nati. Acad. Sci. USA Vol. 85, pp. 7551-7555, October 1988

Biochemistry

Human ,f2-adrenergic receptors expressed in Escherichia coli membranes retain their pharmacological properties (Agtll expression vector/gene fusion)

STEFANO MARULLO, COLETTE DELAVIER-KLUTCHKO, YUVAL ESHDAT*, A. DONNY STROSBERGt, AND LAURENT EMORINE Laboratoire de Biologie Moldculaire des Rdcepteurs, Centre National de la Recherche Scientifique, Universitd Paris VII, and Institut Pasteur, 28 Rue du Dr. Roux 75724, Paris Cedex 15, France

Communicated by Jonathan Beckwith, July 14, 1988 (received for review May 9, 1988)

ABSTRACT

The coding region of the gene for the human

f2-adrenergic receptor gene was fused to the .-galactosidase gene of the Agtll expression vector. The Y1089 Escherichia coil strain was lysogenized with this modified vector and transcription of the fusion gene was induced. Expression of this transcription unit was shown by the appearance in the bacteria of proteins of molecular weight higher than that of native 8-galactosidase, which are immunoreactive with anti-flgalactosidase antibodies. Production of P2-adrenergic receptors was shown by the presence, on intact bacteria, of binding sites for catecholamine agonists and antagonists possessing a typical (2-adrenergic pharmacological profile. Binding and photoaffinity labeling studies performed on intact E. coil and its membrane fractions showed that these binding sites are located in the inner membrane of the bacteria. Expression of pharmacologically active human ,f2-adrenergic receptors in E. coli further supports the similar transmembrane organization proposed for bacteriorhodopsin and eukaryotic membraneembedded receptors coupled to guanine nucleotide-binding regulatory proteins. Moreover, this system should facilitate future analyses of the ligand-binding properties within this family of membrane receptors.

membrane a protein that displays full agonist and antagonist binding activity. These results suggest that the membrane environment required by hu,82AR for ligand binding is conserved in bacteria and that bacterial and eukaryotic membrane embedded receptors might indeed share a common transmembrane organization. Moreover, this expression system will provide a convenient tool for characterization of members of this family of membrane receptors as well as for structure-function relationship studies.

MATERIALS AND METHODS Gene Fusion. An EcoRI to Nco I synthetic linker was ligated to the purified 1.3-kilobase Nco I-Dra I DNA fragment containing the coding region of the huf32AR (3). After phenol extraction and isopropanol precipitation, EcoRI linkers were ligated to this construct which, after EcoRP digestion and subsequent purification, was inserted in the unique EcoRI restriction site of the Agtll vector (Fig. 1). The initiation codon of the hu,82AR is included in the Nco I restriction site and its termination codon is located 19 base pairs on the 5' side of the Dra I restriction site. The synthetic EcoRP-Nco I linker maintains the reading frame of the P-galactosidase gene throughout the hu,32AR gene and contains a sequence coding for an Asp-Pro peptide bond created to facilitate separation of the two fused proteins after mild acid hydrolysis. Constructs were verified for preservation of the EcoRI and Nco I restriction sites and for orientation of the insert. Binding Assays. E. coli Y1089 cells expressing the fusion gene were obtained by standard protocols (11). The cells were harvested and resuspended in a solution of 10 mM Tris HCl (pH 7.4) and 90 mM NaCl. Aliquots containing 6 x 107 cells were incubated for 1 hr at 370C in a final volume of 1 ml with [1251]iodocyanopindolol (ICYP; 2080 Ci/mmol; 1 Ci = 37 GBq; Amersham) at various concentrations (from 1 to 80 pM). Reactions were stopped by filtration on Whatman GF-F filters followed by four rapid washes with 4 ml of buffer. Nonspecific binding was determined in the presence of 1 ,uM (-)-propranolol and was 10 times better than the dextrorotatory isomer, (+)-isoproterenol. Moreover, these binding sites displayed a marked 82adrenergic specificity, as shown by the characteristic order of potencies for p2 ligands over p1 ligands (Table 1). When

Proc. Natl. Acad. Sci. USA 85 (1988)

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Table 1. Binding of 13-adrenergic ligands to hu,32AR expressed in

intact E. coli Pharmacological property Compound Ki, nM 9100 ± 1100 Agonist (-)-Norepinephrine

205 116 97 66 -

4700 ± 1400 Agonist (-)-Epinephrine 1400 ± 100 Agonist (+ )-Isoproterenol 110 ± 10 Agonist (- )-Isoproterenol 8 40 ± Agonist (J32) Procaterol 2500 + 60 Antagonist (i31) CGP 20712-A 790 ± 220 Antagonist (13k) Practolol 1.3 4.8 ± Antagonist CGP 12.177 Each competitor was tested in two to four independent experiments. Stereoisomers are indicated by (+) or (-).

45-

29 -

FIG. 2. Immunoblots with anti-p-galactosidase antibodies. Bacteria (10' lysed cells corresponding to 70 Ag of protein) infected with Agtll phages containing hu/2AR gene in the correct orientation (lane 1), wild-type Agtll (lane 2), and Agtll containing the receptor gene in the opposite orientation (lane 3). Inner membrane protein (40 ,&g, lane 4), and 140 ,ug of outer membrane protein (lane 5) from the bacteria analyzed in lane 1. The following quantities of ,Bgalactosidase were used to form a standard curve: 0.016 (lane 6), 0.04 (lane 7), and 0.1 (lane 8) ,g. For evaluation of immunoreactive material, a transparency of the blot was scanned densitometrically, and the integration of the area under the peaks was compared to the calibration curve of ,8-galactosidase.

binding experiments were performed with bacteria infected with either wild-type Agtll, or a phage containing the hup82AR gene inserted in the opposite direction,