CLONING OF A PUTATIVE G-PROTEIN-COUPLED RECEPTOR FROM ARABIDOPSIS-THALIANA

We have cloned and characterized a cDNA from Arabidopsis thaliana that most likely encodes a novel member of the vast superfamily of G-prote in-coupled receptor proteins (GPCRs). By taking advantage of amino aci d sequence similarities between plant expressed sequence tags (ESTs) a nd established G-protein-coupled receptor sequences, a probe was obtai ned which was used for the screening of an Arabidopsis cDNA library. T he cDNA which was found is very infrequently represented in the cDNA l ibrary, suggesting a low and/or spatially restricted expression. A reg ion of the translated sequence of the cDNA shows the highest similarit y to cAMP receptors from the slime mold Dictyostelium discoideum. The same region is also similar to that in members of the anima. calcitoni n family of receptors. Another region of the putative receptor, howeve r, is similar to sequences of serotonin receptors and other receptors of the so-called rhodopsin family of GPCRs. The rhodopsin family has n umerous members in higher vertebrate species. Alignments and phylogene tic analyses of the regions of similarity yielded results in accordanc e with other evolutionary considerations. Our cDNA thus occurred on a distinct major branch in relation to the rest of the rhodopsin family. In relation to the: calcitonin family, our cDNA and cAMP receptors oc curred together on a distinct major branch but appear to have diverged from each other shortly after their divergence from the rest of the c alcitonin family. Other features further argue for a tentative identif ication of it as a GPCR. It displays seven discrete and strongly predi cted transmembrane domains when analyzed in hydropathy plots. The pref erred orientation is with the amino terminus towards the outside. It h as one Cys residue in extracellular loop 1 and another in extracellula r loop 2. Cys residues in these loops are known to form disulfide brid ges in many other GPCRs. Finally, it has several fully conserved amino acids that belong to the most conserved in previously known GPCRs, th at occur in the above regions of similarity.