Identification and characterization of a pheromone 2 specific binding protein of Euplotes octocarinatus.

Ciliates show two forms of reproduction: asexual (vegetative) and sexual reproduction (conjugation). Conjugation in Euplotes octocarinatus is induced by pheromones secreted by cells of one mating type binding to corresponding receptors on cells of a different mating type. In this study, we used heterologously-expressed pheromone 2 (Phr2) in affinity chromatography to isolate pheromone 2 binding proteins in Euplotes octocarinatus syngen 1. Using this approach, we isolated a 42-kDa protein (Phr2B) that bound Phr2. A tryptic fragment of Phr2B was used to construct degenerate oligonucleotides for PCR amplification of the macronuclear gene. The gene is predicted to encode a 58 amino acid protein. The N-terminus of the deduced protein shows significant similarity to membrane bound protein kinases of such diverse organisms as Xenopus laevis, Mus musculus and Homo sapiens, while the C-terminus contains a region which is similar to an extracellular region within chemosensory receptors of Caenorhabditis elegans. Western blotting analysis demonstrated that Phr2B specifically recognizes Phr2 and preliminary results indicate that antibodies raised against a C-terminal peptide of Phr2B inhibit cell mating. Further studies should be undertaken to investigate the precise role that Phr2B plays in preconjugative intercellular communication in E. octocarinatus. [Copyright &y& Elsevier]