IDENTIFICATION AND INITIAL CHARACTERIZATION OF AN AUTOCRINE PHEROMONE RECEPTOR IN THE PROTOZOAN CILIATE EUPLOTES-RAIKOVI

The polypeptide pheromone Er-1, purified from the ciliate Euplotes raikovi of mating type I and genotype mat-1/mat-1, was iodinated with 125I-Bolton-Hunter reagent to a sp act of 0.45-0.73 μCi/μg of protein. This preparation of 125I-Er-1 bound specifically to high affinity binding sites on the same cells of mating type I. Binding of 125I-Er-1 occurred with an apparent K(d) of 4.63 ± 0.12 x 10-9 M in cells in early stationary phase. It was estimated that these cells carry a total number of ~5 x 107 sites/cell, with a site density that falls in the range of 1,600-1,700/μm2 of cell surface. Unlabeled Er-1, other homologous pheromones such as Er-2 and Er-10, antibodies specific for Er-1, and human IL-2 were shown to act as effective inhibitors of specific binding of 125I-Er-1 to mating type I cells. The 'autocrine' nature of the identified specific high affinity binding sites for Er-1 was further substantiated by cross-linking experiments. These experiments revealed that mating type-I cell membranes contain one protein entity of M(r) = 28,000 that is capable of reacting specifically with the homodimeric native form of Er-1.