A putative b 2 -adrenoceptor from the rainbow trout ( Oncorhynuchus mykiss ) Molecular characterization and pharmacology James G. Nickerson, Stephen G. Dugan, Guy Drouin and Thomas W. Moon Department of Biology, University of Ottawa, Ontario, Canada Extensivemolecularcharacterizationof mammalian b-adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT-PCR approach we have cloned a rainbow troutb 2 -adrenoceptor genethat codesfor a 409-amino-acid protein with the same seven transmembrane domainstructureasitsmammaliancounterparts. This rainbow trout b 2 -adrenoceptor sharesa high degreeof amino-acid sequence conservation with other vertebrate b 2 -adrenoceptors. The conclusion that this sequence is a rainbow troutb 2 -adrenoceptor isfurthersupportedby phylogeneticanalysis of vertebrateb-adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout b 2 -adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G-protein- bindingdomainof the rainbow troutb 2 -adrenoceptor sequencesuggeststhat the in vivo control of trout b 2 -adrenoceptor signaling differs substantially from that of mammals. Keywords: trout; b 2 -adrenoceptor; sequencing; expression; kinetic analysis. The b-adrenoceptors (b-ARs) are members of a family of seventransmembrane domainreceptorscoupledto G-proteinsthat transducethe cellulareffectsof the stresshormonesadrenaline and noradenaline [1,2]. The typical b-AR signal transductionpathwayresultsin increasedcellularcAMP levels causedby G s -protein- mediated stimulation of adenylate cyclase activity [3,4]. The second messenger, cAMP, regulates the activity of severalkey enzymes, mostnotablyproteinkinaseA, which modulate the activity of a numberof proteins within the cell to bring about a tissue specific response [3,5]. The various tissue responseselicited by catecholamine release are aimed at allowing organisms to cope with environmental and physiological stressors [6 – 8]. Due to the implication of the b-ARs in several human disorders such as heart disease and obesity [8 – 11], the role of b-ARs in the stress response has been well characterized in severalmammalian tissues. Increased cardiac output, elevatedblood glucose,increasedthermogenesis in brown adipose tissue and vasodilation of systemic blood vesselsare someof the common adrenergic responses typically associatedwith activationof b-ARs in mammals [4,12]. Pharmacologicaland molecular biologicalstudies in mammals indicate the existence of threeclosely related genes coding forb 1,2 - and 3 -AR subtypes [1,13]. Characterization of the b-ARs in tissuesof early branching vertebrates, such as fish, has occurred predomi- nantly at the pharmacological level. With the exception of Xenopus and turkey [14 – 16], no studies have dealt with the molecular biology of nonmammalian b-ARs. Physiology- based studies using several speciesoffishesrevealthat some ofthe typicalmammalian b-adrenergic responses includingincreasedcardiacoutputand elevated blood glucose levels also occur in fish through agonist mediated b-AR signaling [7,17]. In addition, fish b-ARs elicitresponses unique to particular species or groups of fish. For example,catecholamine releasecausesan increasein hemoglobin/oxygen bindingaffinityin the rainbow trout erythrocyte by activation of a red blood cell b-AR [18]. In spite of the many studies dealing with the physiology and pharmacology of fish b-ARs, manyfundamental questions remain concerning the number of b -AR genes in fishes,the pattern of expression of these genes, and theirregulation. To address some of these questions we havecloned aputativeb 2 -AR from the rainbow trout (Oncorhynchus mykiss ) and studied its expression pattern in eighttissues. This information is the first step towards a broader understanding of fish b-AR systems and provides Note: a web site is available at http://www.uottawa.ca Note: the rainbow trout b 2 -AR sequence reported here has been submitted to the GenBank database and will be available at the time of publication under accession number AY044093 Correspondence to T. W. Moon, Department of Biology, University of Ottawa, 30 Marie Curie Street, PO Box 450, Station A Ottawa, ON, Canada, K1N 6 N5. Fax: þ 1 613 562 5486, Tel.: þ 1 613 562 5800 ext. 6002, E-mail: tmoon@science.uottawa.ca (Received 24 July 2001, revised 5 October 2001, accepted 16 October 2001) Abbreviations: AR, adrenoceptor; GSP, gene specific primer; AUAP, abridged universal amplification primer; CGP-12177, (( – )- 4-(3-t-butylamino-2-hydroxypropoxy-[5,7- 3 H]benzimidazol-2-one); ICI, ICI 118551. Eur. J. Biochem. 268, 6465–6472 (2001) q FEBS 2001