Constitutively Active G Protein-Coupled Receptor Mutants Block Dictyostelium Development

Minghang Zhang,* Mousumi Goswami, and Dale Hereld ${dagger}$

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

Monitoring Editor: Peter Devreotes

cAR1, a G protein-coupledreceptor (GPCR) for cAMP, is required for the multicellulardevelopment of Dictyostelium. The activation of multiple pathwaysby cAR1 is transient due to poorly defined adaptation mechanisms.To investigate this, we used a genetic screen for impaired developmentto isolate four dominant-negative cAR1 mutants, designated DN1-4.The mutant receptors inhibit multiple cAR1-mediated responsesknown to undergo adaptation. Reduced in vitro adenylyl cyclaseactivation by GTP ${gamma}$ S suggests that they cause constitutive adaptationof this and perhaps other pathways. In addition, the DN mutantsare constitutively phosphorylated, which normally requires cAMPbinding, and possess cAMP affinities that are $~$ 100-fold higherthan that of wild-type cAR1. Two independent activating mutations,L100H and I104N, were identified. These residues occupy adjacentpositions near the cytoplasmic end of the receptor’s thirdtransmembrane helix and correspond to the (E/D)RY motif of numerousmammalian GPCRs, which is believed to regulate their activation.Taken together, these findings suggest that the DN mutants areconstitutively activated and block development by turning onnatural adaptation mechanisms.

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

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