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The cyclic nucleotide specificity of three cAMP receptors in Dictyostelium
R L Johnson1, P J Van Haastert, A R Kimmel
1Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205.
Abstract:
cAMP receptors mediate signal transduction pathways during development in Dictyostelium. A cAMP receptor (cAR1) has been cloned and sequenced (Klein, P., Sun, T. J., Saxe, C. L., Kimmel, A. R., Johnson, R. L., and Devreotes, P. N. (1988) Science 241, 1467-1472) and recently several other cAR genes have been identified (Saxe, C. L., Johnson, R., Devreotes, P. N., and Kimmel, A. R. (1991a) Dev. Genet. 12, 6-13; Saxe, C. L., Johnson, R. L., Devreotes, P. N., and Kimmel, A. R. (1991b) Genes Dev. 5, 1-8). We have expressed three receptor subtypes, cAR1, cAR2, and cAR3, in growing cells and have investigated their affinity and pharmacological specificity in a series of [3H]cAMP binding studies. In phosphate buffer, there were two affinity states of about 30 and 300 nM for cAR1 and 20 and 500 nM for cAR3 but no detectable affinity for cAR2. In the presence of 3 M ammonium sulfate, there was one affinity state of 4 nM for cAR1 and 11 nM for cAR2 and two affinity states of approximately 4 and 200 nM for cAR3. The relative affinities of 14 cyclic nucleotide derivatives were tested for each cAR in ammonium sulfate. These studies suggest a model (Van Haastert, P. J. M., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) in which cAMP binds to all three receptor subtypes by maintaining hydrogen bond interactions at the N6 and O3' positions. Interactions at the exocyclic oxygens of cAMP varied between the receptors; cAR2 and cAR3 lacked a stereoselective interaction at the axial oxygen which was present in cAR1. The cleft, which binds the adenine ring of cAMP, was hydrophobic in cAR1 and cAR3 but relatively polar in cAR2. The analog specificity of cAR1 and cAR3 in phosphate buffer was similar to that measured in ammonium sulfate though the derivatives' relative affinity to cAMP was reduced. We conclude that these cAMP receptor subtypes can be distinguished by distinct pharmacological properties which will allow selective activation of each cAR during development.
Insights
Cyclic adenosine monophosphate (cAMP) receptors are crucial for Dictyostelium development. This study characterizes three cAMP receptor subtypes (cAR1, cAR2, cAR3), revealing distinct pharmacological properties that enable selective activation during development.
Area of Science:
- Cellular and Molecular Biology
- Developmental Biology
- Biochemistry
Background:
- Cyclic adenosine monophosphate (cAMP) receptors are essential for signal transduction during Dictyostelium development.
- Previous work identified the cAR1 receptor and recently several other cAMP receptor (cAR) genes.
Purpose of the Study:
- To express and characterize the affinity and pharmacological specificity of three cAMP receptor subtypes: cAR1, cAR2, and cAR3.
- To investigate the binding interactions of various cyclic nucleotide derivatives with these receptor subtypes.
Main Methods:
- Expression of cAR1, cAR2, and cAR3 in growing Dictyostelium cells.
- [3H]cAMP binding studies were performed in phosphate buffer and in the presence of 3 M ammonium sulfate.
- Relative affinities of 14 cyclic nucleotide derivatives were tested for each cAR in ammonium sulfate.
Main Results:
- Distinct affinity states were observed for cAR1 and cAR3 in phosphate buffer, while cAR2 showed no detectable affinity.
- In ammonium sulfate, cAR1 and cAR2 exhibited single affinity states, whereas cAR3 showed two affinity states.
- Analysis of cyclic nucleotide derivative binding revealed specific hydrogen bond interactions and variations in adenine ring binding cleft hydrophobicity among the receptor subtypes.
Conclusions:
- The three cAMP receptor subtypes (cAR1, cAR2, cAR3) possess distinct pharmacological properties.
- These differences allow for the selective activation of each cAMP receptor subtype during Dictyostelium development.