Folate chemotactic receptors in Dictyostelium discoideum II. Guanine nucleotides alter the rates of interconversion and the proportioning of four receptor states

doi:10.1016/0167-4889(86)90214-4

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Volume 886, Issue 1, 8 April 1986, Pages 88-95

https://doi.org/10.1016/0167-4889(86)90214-4 Get rights and content

Abstract

The ligand binding properties of folate chemotactic receptors on isolated membranes of Dictyostelium discoideum were analyzed. Three out of the four receptor states (B^F, B^S and B^SS) were detected, showing rate constants and K_d values similar to those obtained for intact cells. Guanine nucleotides changed the proportioning of the receptor states as well as the rates of several conversions. (i) The transformation of B^F into B^S was inhibited by GDP but not by guanylyl imidodiphosphate (GuaPP[NH]P) or GTP. (ii) The number of B^S sites was lowered by GTP and GuaPP[NH]P. (iii) The binding to B^SS was lowered by GTP and GDP, but increased by GuaPP[NH]P. (iv) The rate of disappearance of B^SS was increased by GTP, but not by GuaPP[NH]P. Effects of guanine nucleotides were not observed after treatment of the membrane preparations with 15 mg/ml bovine serum albumin. This treatment caused the detection of a binding type different from the types described previously. The affinity of this binding site was extremely high (K_d ≤ 0.2 nM for N₁₀-methylfolic acid), while the dissociation was relatively slow (k₋₁ ≤ 3·10⁻⁴ s⁻¹). It is proposed that bovine serum albumin uncouples the folate receptor from a guanine nucleotide regulatory (G) protein in an irreversible manner. A model is presented in which the four receptor states correspond to distinct interactions with a G protein and GDP or GTP.

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Cited by (14)

Acanthamoeba and Dictyostelium Use Different Foraging Strategies
2016, Protist
Citation Excerpt :
Signaling through these cAMP receptors continues during the multicellular phases of development and contributes to cell sorting within the aggregate and the differentiation of cells into the stalk or spores of the fruiting body (Kim et al. 1998; Louis et al. 1994; Saxe et al. 1993). In contrast, chemotactic movement associated with foraging is present during vegetative growth and enhanced during the first few hours of starvation (De Wit and Bulgakov 1986; Hadwiger et al. 1994; Pan et al. 1972, 1975). This chemotactic response requires receptors for pterin-like compounds such as folate.
Amoeba often use cell movement as a mechanism to find food, such as bacteria, in their environment. The chemotactic movement of the soil amoeba Dictyostelium to folate or other pterin compounds released by bacteria is a well-documented foraging mechanism. Acanthamoeba can also feed on bacteria but relatively little is known about the mechanism(s) by which this amoeba locates bacteria. Acanthamoeba movement in the presence of folate or bacteria was analyzed in above agar assays and compared to that observed for Dictyostelium. The overall mobility of Acanthamoeba was robust like that of Dictyostelium but Acanthamoeba did not display a chemotactic response to folate. In the presence of bacteria, Acanthamoeba only showed a marginal bias in directed movement whereas Dictyostelium displayed a strong chemotactic response. A comparison of genomes revealed that Acanthamoeba and Dictyostelium share some similarities in G protein signaling components but that specific G proteins used in Dictyostelium chemotactic responses were not present in current Acanthamoeba genome sequence data. The results of this study suggest that Acanthamoeba does not use chemotaxis as the primary mechanism to find bacterial food sources and that the chemotactic responses of Dictyostelium to bacteria may have co-evolved with chemotactic responses that facilitate multicellular development.
Activation of the mitogen-activated protein kinase ERK2 by the chemoattractant folic acid in Dictyostelium
1997, Journal of Biological Chemistry
The Dictyostelium MAP kinase ERK2 is activated by extracellular cAMP in aggregation-competent cells and is required for receptor activation of adenylyl cyclase (Maeda, M., Aubry, L., Insall, R., Gaskins, C., Devreotes, P. N., and Firtel, R. A. (1996) J. Biol. Chem. 271, 3351–3354; Segall, J., Kuspa, A., Shaulsky, G., Ecke, M., Maeda, M., Gaskins, C., Firtel, R., and Loomis, W. (1995) J. Cell Biol. 128, 405–413). This cAMP-dependent activation of ERK2 is mediated by the serpentine, G protein-coupled cAMP receptors. However, ERK2 activation by cAMP is at least partially heterotrimeric G protein-independent, with a level of activation in cells lacking the sole Gβ subunit or the G protein-coupled cAMP receptors-coupled Gα2 subunit that is ∼50% that of wild-type cells (Maeda, M., Aubry, L., Insall, R., Gaskins, C., Devreotes, P. N., and Firtel, R. A. (1996)J. Biol. Chem. 271, 3351–3354; Segall, J., Kuspa, A., Shaulsky, G., Ecke, M., Maeda, M., Gaskins, C., Firtel, R., and Loomis, W. (1995) J. Cell Biol. 128, 405–413). Folic acid, a chemoattractant in the vegetative cells that enables amoebae to find bacteria in the wild, also triggers the activation of adenylyl cyclase, which is impaired in the vegetative cells lacking the Gα protein subunit Gα4 (Hadwiger, J., Lee, S., and Firtel, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10566–10570). In this study, we show that folic acid activates ERK2 in developmentally regulated manner and is required for ERK2 stimulation of adenylyl cyclase activity. Maximum levels of folate-stimulated ERK2 activity occur in cells from very early in development, prior to aggregation, and again at the tipped aggregate stages, corresponding to the stages in which folate receptors and the coupled Gα subunit Gα4 are maximally expressed. During the activation by folic acid, ERK2 is phosphorylated on tyrosine residue(s) and contemporaneously shows a mobility shift on SDS-PAGE. Interestingly, this activation is not elicited in the absence of Gβ subunits, in contrast to the response to cAMP. This response also requires the Gα4 subunit known to be required for other folic acid-mediated responses (Hadwiger, J., Lee, S., and Firtel, R. (1994)Proc. Natl. Acad. Sci. U. S. A. 91, 10566–10570). Furthermore, we show that the activation of ERK2 by cAMP is independent of the Gα4 subunit, while the activation of ERK2 by folate is independent of Gα2. Taken together, these data indicate that there are at least two pathways of ERK2 activation, heterotrimeric G protein-dependent and -independent pathways.
Dictyostelium transiently expresses eight distinct G-protein α-subunits during its developmental program
1991, Biochemical and Biophysical Research Communications
Guanine nucleotide-binding proteins (G-proteins) play important roles in signal transduction and in controlling the developmental program of Dictyostelium discoideum. While five G-protein α-subunits have been identified in this organism, several lines of evidence suggest that additional G-proteins exist. Using the polymerase chain reaction technique, we have isolated three novel G-protein α-subunit genes. The predicted amino acid sequences of these PCR fragments are very similar to other Dictyostelium G-protein α-subunits in the conserved regions and share little homology outside of those regions. Northern blot analyses indicate that each of these genes has very distinct pattern of expression during development. The possible roles of these G-protein α-subunits are discussed.
G protein linked signal transduction pathways in development: Dictyostelium as an experimental system
1989, Cell
Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum
1988, Experimental Cell Research
The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-γ-l-glutamyl-l-ornithine-δ-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with K_d values in the range of 20 and 100 nM. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (<1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 °C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 °C. Two glorinase activities were observed. An ornithine δ-lactam cleaving activity with a K_m of ca. 10⁻⁴M and a propionic acid removing activity (K_m 10⁻⁵M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10–100 nM range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTPγS binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.
The evolutionary origin of eukaryotic transmembrane signal transduction
1988, Comparative Biochemistry and Physiology -- Part A: Physiology
- 1.
  1. A comparison was made of transmembrane signal transduction mechanisms in different eukaryotes and prokaryotes.
- 2.
  2. Much attention was given to eukaryotic microbes and their signal transduction mechanisms, since these organisms are intermediate in complexity between animals, plants and bacteria.
- 3.
  3. Signal transduction mechanisms in eukaryotic microbes, however, do not appear to be intermediate between those in animals, plants and bacteria, but show features characteristic of the higher eukaryotes.
- 4.
  4. These similarities include the regulation of receptor function, adenylate cyclase activity, the presence of a phosphatidylinositol cycle and of GTP-binding regulatory proteins.
- 5.
  5. It is proposed that the signal transduction systems known to operate in present-day eukaryotes evolved in the earliest eukaryotic cells.

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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Regular paperFolate chemotactic receptors in Dictyostelium discoideum II. Guanine nucleotides alter the rates of interconversion and the proportioning of four receptor states

Abstract

Differentiation

FEBS Lett.

Biochem. Biophys. Res. Commun.

Dev. Biol.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Cell

J. Biol. Chem.

Biochim. Biophys. Acta

FEBS Lett.

Biochem. Biophys. Res. Commun.

Exp. Cell Res.

Biochem. Biophys. Res. Commun.

Acanthamoeba and Dictyostelium Use Different Foraging Strategies

Activation of the mitogen-activated protein kinase ERK2 by the chemoattractant folic acid in Dictyostelium

Dictyostelium transiently expresses eight distinct G-protein α-subunits during its developmental program

G protein linked signal transduction pathways in development: Dictyostelium as an experimental system

Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum

The evolutionary origin of eukaryotic transmembrane signal transduction

Regular paper
Folate chemotactic receptors in Dictyostelium discoideum II. Guanine nucleotides alter the rates of interconversion and the proportioning of four receptor states