EXPRESSION OF BETA(2)-ADRENOCEPTORS IN HALOBACTERIA

Halobacteria are halophilic representatives of the recently defined do main, the Archaea. Halobacterium salinarium belongs to this group of m icroorganisms and contains large amounts of bacteriorhodopsin in its m embrane. Bacteriorhodopsin is a seven-transmembrane protein that consi sts of bacterio-opsin (BO), and the chromophore retinal, which is cova lently attached to BO. We have investigated whether the expression mac hinery for BO can be utilized for synthesis of the human beta(2)-adren oceptor (beta(2)-AR), a protein with a similar seven-transmembrane-hel ix topology. An expression vector for BO synthesis was modified to exp ress beta(2)-ARs under the control of BO regulatory elements in H. sal inarium. Homologous recombination into the genome was verified by poly merase chain reactions. Northern blots revealed transcripts of the cal culated size and significant amounts of epitope-tagged beta(2)-ARs wer e detected in Western blots. However, binding of the beta-AR antagonis t I-125-cyanopindolol revealed low levels of functional receptors, and the ligand binding properties of these receptors were altered when co mpared to native receptors. Expression of chimeras containing larger a mino terminal portions of BO did not result in higher receptor levels. Expression of beta(2)-AR in Haloferax volcanii, another member of hal obacteria, was achieved with a vector carrying the ferredoxin promoter . The levels of functional receptor as determined by I-125-cyanopindol ol binding were 180 fmol/mg protein. The beta-AR ligands isoprenaline and propranolol showed affinities expected for functional beta(2)-ARs. Thus, functional human beta(2)-ARs were expressed in halobacteria, co nstituting a first approach for expression of a eukaryotic protein in the domain of Archaea.