Autocrine, Mitogenic Pheromone Receptor Loop of the Ciliate Euplotes raikovi : Pheromone-Induced Receptor Internalization

Abstract

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.In association with the evolution of systems of multiple cell (mating) types controlling self/nonself recognition phenomena, species of Euplotes synthesize families of structurally homologous soluble proteins that confer on cells a chemical specificity, with each protein representing a diffusible chemical signal (pheromone) that distinguishes one cell type from all the others (17). Euplotes raikovi is the species with which these pheromones, designated Er-1, Er-2, and so forth, have been better characterized with regard to their structure and function. Pheromones are small molecules of 38 to 50 amino acids with a common architecture based on a bundle of three ␣-helices fastened together by three conserved disulfide bonds (16,26,35). They have at least two activities each, acting as both prototypic autocrine growth factors and paracrine mating (sexual) signals (31). Upon binding their own pheromones, which cells constitutively secrete into the extracellular environment throughout the cell cycle, cells grow vegetatively and divide mitotically. When cells bind a nonself pheromone from another cell type, they temporarily arrest their growth and develop competence for uniting in mating pairs (23). We refer to the response to self pheromones as an autocrine response and the response to nonself pheromones as a paracrine response.The field addressing the issue of how cells can discriminate between autocrine and paracrine pheromone binding and accordingly mount a growth or mating response was advanced by the identification of the pher...

Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii

“…This peculiar organization of the E. nobilii pheromone genes probably reflects a conserved inclusion of intron sequences, whose splicing might have a fundamental role in the mechanism of expression of these genes. This hypothesis receives support in particular from previous functional analysis of E. raikovi pheromone genes [26,27], showing that the primary transcripts of these genes undergo intron splicing (at not canonical sites) to generate membranebound pheromone isoforms that cells use as autocrine pheromone receptors [9]. Its assessment, in addition to being crucial to elucidate the mechanism of expression of the E. nobilii pheromone gene family, may also contribute insightful information on the more general problem of the functional organization of the sub-chromosomic, gene-sized macronuclear genomes of the hypotrich ciliates.…”

“…As in other species of Euplotes [6], these mating types appear to be under the control of a series of alleles co-dominant at the Mendelian locus mat of the chromosomic genome (transcriptionally silent) of germ-line nucleus (micronucleus) [7, and unpublished results], and become manifest through the activity of specific and diffusible signal proteins usually referred to as "pheromones". Cells grow in response to the autocrine (homologous) binding of their secreted (self) pheromones, and temporarily arrest their growth and unite into mating pairs in response to the paracrine (heterologous) binding of foreign (non-self) pheromones [8,9].…”

Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating

“…Results from studies on E. raikovi (24) suggest that the pheromone receptors are membrane-bound pheromone isoforms, characterized in each cell type by an extracellular domain that is a structural counterpart to the secreted pheromone capable of selective, competitive binding to multiple closely homologous pheromone molecules (25). Mutual binding reactions between En-6 and En-A1 pheromone preparations were analyzed with Surface Plasmon Resonance experiments, by linking one preparation to a sensor chip to act as a ligand and injecting the second preparation into the flow chamber of the experimental apparatus to act as the analyte (Fig.…”

Fungal Sex and Pathogenesis

“…In the fission yeast Schizosaccharomyces pombe, expressing the M-factor receptor (one of the mating pheromone receptors) in cells that normally express the P-factor receptor results in activation of the mating response, although not to the extent that mating is observed (128). Pheromones in the ciliate Euplotes raikovi and the fungus Cryptococcus neoformans act both in an autocrine manner (to promote growth and differentiation) and also in a paracrine manner (as a sexual pheromone) (251). In basidiomycetes like Cr.…”