A Single Cell Density-sensing Factor Stimulates Distinct Signal Transduction Pathways through Two Different Receptors

William J Deery,Tong Gao,Robin Ammann,Richard H Gomer

doi:10.1074/jbc.m204539200

William J Deery, Tong Gao + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m204539200

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Abstract

In Dictyostelium discoideum, cell density is monitored by levels of a secreted protein, conditioned medium factor (CMF). CMFR1 is a putative CMF receptor necessary for CMF-induced G protein-independent accumulation of the SP70 prespore protein but not for CMF-induced G protein-dependent inositol 1,4,5-trisphosphate production. Using recombinant fragments of CMF, we find that stimulation of the inositol 1,4,5-trisphosphate pathway requires amino acids 170-180, whereas SP70 accumulation does not, corroborating a two-receptor model. Cells lacking CMFR1 do not aggregate, due to the lack of expression of several important early developmentally regulated genes, including gp80. Although many aspects of early developmental cAMP-stimulated signal transduction are mediated by CMF, CMFR1 is not essential for cAMP-stimulated cAMP and cGMP production or Ca(2+) uptake, suggesting the involvement of a second CMF receptor. Exogenous application of antibodies against either the region between a first and second or a second and third possible transmembrane domain of CMFR1 induces SP70 accumulation. Antibody- and CMF-induced gene expression can be inhibited by recombinant CMFR1 corresponding to the region between the first and third potential transmembrane domains, indicating that this region is extracellular and probably contains the CMF binding site. These observations support a model where a one- or two-transmembrane CMFR1 regulates gene expression and a G protein-coupled CMF receptor mediates cAR1 signal transduction.

Highlights

Little is known about how specific cell types within a tissue population are able to sense their cell density
We previously identified a putative receptor for conditioned medium factor (CMF), CMFR1, which is necessary for CMF-stimulation of G protein-independent prestalk and prespore gene expression but not G protein-dependent IP3 production [40]
The KD for both cell types was what we previously observed for wild-type cells [10, 40], whereas the number of CMF receptors for Ax2 cells was considerably less than that reported for Ax4

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Vegetative Dictyostelium Ax2 wild-type parental and cmfr1Ϫ cells were grown in suspension shaking culture with HL-5, antibiotics, and vitamins as described previously [41]. Precipitated CMF protein fragments were collected by centrifugation at 20,000 ϫ g for 15 min, resuspended in 20 mM Tris-HCl, pH 7.5, and purified using the B-PER His spin purification kit. IP3, cAMP, and cGMP Production and Ca2ϩ Uptake—The production of intracellular IP3 in response to a 30-s pulse of CMF was determined following the procedure of Van Haastert [46] using an IP3 3H assay system (Amersham Biosciences) to quantitate IP3 with the exception that 100-␮l aliquots of neutralized cell supernatants were used. The production of cGMP in response to a 10-s pulse of 0.1 ␮M cAMP was determined in a similar manner following the procedure of Kesbeke et al [47], using a cGMP assay kit (Amersham Biosciences).

TABLE I Binding of CMF to cells

RESULTS

With cAMP pulses

With cAMP

DISCUSSION