Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Nov 4;286(44):38649-38658.
doi: 10.1074/jbc.M111.284182. Epub 2011 Sep 12.

Direct biochemical measurements of signal relay during Dictyostelium development

Satarupa Das  1 , Erin C Rericha  2 , Anna Bagorda  1 , Carole A Parent  3
Affiliations

Direct biochemical measurements of signal relay during Dictyostelium development

Satarupa Das et al. J Biol Chem. .

Abstract

Upon starvation, individual Dictyostelium discoideum cells enter a developmental program that leads to collective migration and the formation of a multicellular organism. The process is mediated by extracellular cAMP binding to the G protein-coupled cAMP receptor 1, which initiates a signaling cascade leading to the activation of adenylyl cyclase A (ACA), the synthesis and secretion of additional cAMP, and an autocrine and paracrine activation loop. The release of cAMP allows neighboring cells to polarize and migrate directionally and form characteristic chains of cells called streams. We now report that cAMP relay can be measured biochemically by assessing ACA, ERK2, and TORC2 activities at successive time points in development after stimulating cells with subsaturating concentrations of cAMP. We also find that the activation profiles of ACA, ERK2, and TORC2 change in the course of development, with later developed cells showing a loss of sensitivity to the relayed signal. We examined mutants in PKA activity that have been associated with precocious development and find that this loss in responsiveness occurs earlier in these mutants. Remarkably, we show that this loss in sensitivity correlates with a switch in migration patterns as cells transition from streams to aggregates. We propose that as cells proceed through development, the cAMP-induced desensitization and down-regulation of cAMP receptor 1 impacts the sensitivities of chemotactic signaling cascades leading to changes in migration patterns.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
The ACA activity measured following subsaturating cAMP stimulations is a biochemical measurement of signal relay. A, ACA activity was measured after the addition of 1 nm or 10 μm cAMP in 5-h developed WT cells. Partially purified PdsA was added to cells 45 s after the initial cAMP stimulus (indicated by the asterisk). The absolute basal ACA activity (pmol/min/mg) value is 2.8 ± 0.6 S.D. (n = 3). B, ACA activity was measured after the addition of subsaturating concentration of the nonhydrolyzable cAMP analog Sp-cAMPS in WT cells. Partially purified PdsA was added to cells 45 s after the initial Sp-cAMPS stimulus (indicated by the asterisk). The absolute basal ACA activity (pmol/min/mg) value is 4.8 ± 0.2 S.D. (n = 3). The results represent the averages (± S.D.) of three independent experiments.
FIGURE 2.
FIGURE 2.
Subsaturating cAMP stimulations reveal developmental time-dependent differences in ACA activation. A, C, and E, ACA activity was measured in 3-h developed WT (A), regA− (C), and pkaR− (E) cells following 1 nm or 10 μm cAMP stimulations. Partially purified PdsA was added to cells 1 min after the initial stimulus (indicated by the asterisk) in each case as described in Fig. 1. The absolute basal ACA activity (pmol/min/mg) values are: 3.2 ± 0.8 S.D. (n = 3) for WT, 4.3 ± 0.9 S.D. (n = 3) for regA−, 4.9 ± 1.1 S.D. (n = 3) for pkaR−. B, D, and F, ACA activity was measured in 5-h developed WT (B), regA− (D), and pkaR− (F) cells following 1 nm or 10 μm cAMP stimulations. In addition, ACA activity was measured in 7-h developed WT cells (B) following a 10 μm cAMP stimulation. The absolute basal ACA activity (pmol/min/mg) values at 5 h are: 3.1 ± 0.6 S.D. (n = 3) for WT, 8.1 ± 1.15 S.D. (n = 3) for regA−, and 9.8 ± 0.9 S.D. (n = 3) for pkaR−, and the value at 7 h for WT is 9.1 ± 1.13 S.D. (n = 1). The results represent the average (± S.D.) of three independent experiments.
FIGURE 3.
FIGURE 3.
In later development, cells display high basal ACA activity and intracellular cAMP levels. A, the absolute basal ACA activity in pmol/min/mg was measured at indicated developmental time points as described in Fig. 1 in the absence of cAMP stimulation. The results represent the averages (± S.D.) of three independent experiments. B, basal FRET efficiency images represented in pseudo-color for 3–7-h developed WT and regA− cells. Percentage basal FRET efficiency was calculated from the FRET efficiency images, and results represent the averages ± S.D. from four independent experiments.
FIGURE 4.
FIGURE 4.
Basal cAR1 phosphorylation increases during Dictyostelium development. cAR1 phosphorylation was measured in 3- or 5-h developed WT (A), regA− (B), and pkaR− (C) cells 15 min after 1 nm or 10 μm cAMP stimulations. In addition, cAR1 phosphorylation was measured in 7-h developed WT cells (A) 15 min after 1 nm or 10 μm cAMP stimulations. The data shown are representative of results from three independent experiments.
FIGURE 5.
FIGURE 5.
Subsaturating stimulations induce a TORC2 relay response. A, C, and E, PKBR1 phosphorylation was used as a readout of TORC2 activity and was measured in 3-h developed WT (A), regA− (C), and pkaR− (E) cells following 1 nm or 10 μm cAMP stimulations. B, D, and F, PKBR1 phosphorylation was measured in 5-h developed WT (B), regA− (D), and pkaR− (F) cells following 1 nm or 10 μm cAMP stimulations. The results represent the averages (± S.D.) of three independent experiments. Also see supplemental Fig. S2 for a typical Western blot.
FIGURE 6.
FIGURE 6.
Subsaturating stimulations induce an ERK2 relay response. A, C, and E, ERK2 phosphorylation was measured in 3 h developed WT (A), regA− (C), and pkaR− (E) cells following 1 nm or 10 μm cAMP stimulations. B, D, and F, ERK2 phosphorylation was measured in 5-h developed WT (B), regA− (D), and pkaR− (F) cells following 1 nm or 10 μm cAMP stimulations. The results represent the averages (± S.D.) of three independent experiments. Also see supplemental Fig. S3 for a typical Western blot.
FIGURE 7.
FIGURE 7.
Schematic depicting the signal transduction events that lead to desensitized responses during the early development of Dictyostelium. See text for details.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Weijer C. J. (2009) J. Cell Sci. 122, 3215–3223 - PubMed
    1. McMains V. C., Liao X. H., Kimmel A. R. (2008) Ageing Res. Rev. 7, 234–248 - PMC - PubMed
    1. Garcia G. L., Parent C. A. (2008) J. Microsc. 231, 529–534 - PMC - PubMed
    1. Swaney K. F., Huang C. H., Devreotes P. N. (2010) Annu. Rev. Biophys. 39, 265–289 - PMC - PubMed
    1. Dinauer M. C., MacKay S. A., Devreotes P. N. (1980) J. Cell Biol. 86, 537–544 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources