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The ciliate Euplotes raikovi produces a family of diffusible signal proteins (pheromones) that function as prototypic growth factors. They may either promote cell growth, by binding to pheromone receptors synthesized by the same cells from which they are secreted (autocrine activity), or induce a temporary cell shift from the growth stage to a mating (sexual) one by binding to pheromone receptors of other, conspecific cells (paracrine activity). In cells constitutively secreting the pheromone Er-1, it was first observed that the expression of the Er-1 receptor "p15," a type II membrane protein of 130 amino acids, is quantitatively correlated with the extracellular concentration of secreted pheromone. p15 expression on the cell surface rapidly and markedly increased after the removal of secreted Er-1 and gradually decreased in parallel with new Er-1 secretion. It was then shown that p15 is internalized through endocytic vesicles following Er-1 binding and that the internalization of p15/Er-1 complexes is specifically blocked by the paracrine p15 binding of Er-2, a pheromone structurally homologous to, and thus capable of fully antagonizing, Er-1. Based on previous findings that the p15 pheromone-binding site is structurally equivalent to Er-1 and that Er-1 molecules polymerize in crystals following a pattern of cooperative interaction, it was proposed that p15/Er-1 complexes are internalized as a consequence of their unique property (not shared by p15/Er-2 complexes) of undergoing clustering.

Cell type-specific protein signals, called pheromones, are constitutively secreted by Euplotes raikovi and bound back in autocrine fashion, with a positive effect on the vegetative (mitotic) cell growth. In cells growing suspended with their secreted pheromone, it was found that any interruption of this autocrine signaling loop was immediately followed by an effective enhancement of the basal intracellular cyclic AMP (cAMP) level. To establish a cause-effect relationship between these pheromone-induced variations in the cytoplasmic cAMP level and cell growth, cells ready to pass from a resting stage to a new growth cycle were conditioned either to incorporate a cAMP analog resistant to phosphodiesterase degradation, or to utilize cAMP released (following cell irradiation) from incorporated "caged" cAMP. Cells responded at every induced increase in their basal cAMP level by markedly decreasing their commitment to start a new growth cycle. It was deduced that the autocrine signaling of E. raikovi pheromones involves cAMP as inhibitor of its mitogenic activity.

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of 125I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10(-9) M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 10(6) per cell of 5-7 fissions of age, to about 16 x 10(6) at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from "immature" to "adult," that is competent to respond as well to pheromones of conspecific, genetically different cells.