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Vol. 16, Issue 2, 562-572, February 2005

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Constitutively Active G Protein-coupled Receptor Mutants Block Dictyostelium Development

Minghang Zhang ^*, Mousumi Goswami, and Dale Hereld 

Department of Microbiology and Molecular Genetics, The University of Texas Medical School at Houston, Houston, TX 77030

Submitted June 7, 2004; Accepted November 16, 2004
Monitoring Editor: Peter Devreotes

cAR1, a G protein-coupled receptor (GPCR) for cAMP, is required for the multicellular development of Dictyostelium. The activation of multiple pathways by cAR1 is transient because of poorly defined adaptation mechanisms. To investigate this, we used a genetic screen for impaired development to isolate four dominant-negative cAR1 mutants, designated DN1-4. The mutant receptors inhibit multiple cAR1-mediated responses known to undergo adaptation. Reduced in vitro adenylyl cyclase activation by GTPS suggests that they cause constitutive adaptation of this and perhaps other pathways. In addition, the DN mutants are constitutively phosphorylated, which normally requires cAMP binding and possess cAMP affinities that are 100-fold higher than that of wild-type cAR1. Two independent activating mutations, L100H and I104N, were identified. These residues occupy adjacent positions near the cytoplasmic end of the receptor's third transmembrane helix and correspond to the (E/D)RY motif of numerous mammalian GPCRs, which is believed to regulate their activation. Taken together, these findings suggest that the DN mutants are constitutively activated and block development by turning on natural adaptation mechanisms.

Abbreviations used: CHAPS, 3-[(cholamidopropyl)-dimethylammonio]-1-propanesulfonate; CRAC, cytosolic regulator of adenylyl cyclase; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; GTPS, guanosine 5'-[-thio]triphosphate; PDE, phosphodiesterase; PH, pleckstrin-homology; PI3K, phosphatidylinositol 3-kinase; PI-PLC, phosphatidylinositol-specific phospholipase C; PKA, protein kinase A; PKB, protein kinase B; TM, transmembrane domain.

^* Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030.

Corresponding author. E-mail address: dhereld{at}uth.tmc.edu.