Cloning of potential candidates for guinea pig opioid receptors

Abstract

The endogenous opioid system plays an important role in modulating endocrine, respiratory, cardiovascular and immune functions and others (1). Opioids exert their actions by binding to specific, G-protein coupled seven-transmembrane domaine receptors, which have been classified by their ligands as delta, mu, and kappa. Based on the recently cloned mouse delta opioid receptor DNA sequence (2,3), other opioid receptors were also successfully cloned (4,5). In light of the recent cloning and pharmacological characterization of the guinea pig (gp) kappa receptor in our lab (4), the aim of this work was to clone and characterize the gp brain deltaand mu receptors. We were able to clone a 675 bp guinea pig mu opioid receptor sequence, corresponding to the AA 153-383 of the rat mu opioid receoptor (5). Additionally we were able to clone a 571 bp fragment, homologous to the AA 138-329 of the mouse delta opioid receptor (1,2). Methods: a) cDNA library construction and screening: we used a Lambda gt11 gp brain cDNA library (Clonetech) with 1.3 million independent clones, oligo(dT)-primed, cDNA's inserted into the EcoRI site of Lambda gt11 and ranging in size from 1.3kb to 4kb. For new libraries we used gp brain mRNAs to construct several cDNA libraries in the following vectors: pME18SNeo, pME18S, pcD-Neo-SRa-X, b) PCPc PI: 5'-CTCACCATGATGAGCG TGGA-3'; P2: 5 ' -AGCAGCGCTFGAAG~CG-3 ' , P3: 5'-TCGATCCACTGTATFAGCCG3'; muPC~ PI+P3, lx [94oc 1.Stain], 30x [56oc 2min, 72oc 2.Smin, 94oc lmin], lx [56oc 2min, 72oc 8min]. deltaPCR: PI+P2, lx [94oc 1.5mini, 30x [60oc 1.5min, 72oc 2min, 94oc 1rain], lx [60oc 1.5min, 72°C 6min]. The resulting PCR fragments were isolated and finally cloned into pBSSKII+ (Stratagene). After transformation into E.coli and colony screening, the plasmids from positive clones were isolated, and both strands were sequenced using the FCR primer and the plasmid T7and T3 RNA polymerase promoter primer. Results: The isolated DNAs from those gp cDNA libraries (including the Lambda gt11 gp brain cDNA library) have been hybridized with a rat delta opioid receptor (470bp, part of the ORF) and a rat mu opioid receptor probe (1.4kb, containing the complete ORF). All cDNA libraries gave positive signals with both probes and were subsequently used for the PCR reactions. Using the sequence analysis software GCG (University Wisconsin) we translated both sequences into open reading frames and aligned them to the rat and guinea pig kappa-, the rat muand the mouse delta opioid receptor sequences. The results are shown in Fig. 1.