PubMed · 10545905
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K Beaumont, P Negulescu. 1999. Chipping away at GPCR function.. https://doi.org/10.1038/15042
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The Ste2 gene encodes the yeast alpha-pheromone receptor that belongs to the superfamily of seven-transmembrane G protein-coupled receptors. Binding of pheromone induces activation of the heterotrimeric G protein triggering growth arrest in G1 phase and induction of genes required for mating. By random PCR-mediated mutagenesis we isolated mutant 8L4, which presents a substitution of an asparagine residue by serine at position 388 of the alpha-factor receptor. The 8L4 mutant strain shows phenotypic defects such as: reduction in growth arrest after pheromone treatment, diminished activation of the Fus1 gene, and impaired mating competence. The asparagine residue lies in the second half of the intracellular protruding C-terminal tail of the receptor, and its replacement by serine affects interaction with both the G(alpha) and Gbeta subunits. Since expression of the receptor as well as its kinetic parameters, i.e., ligand affinity and receptor number, are unaffected in the mutant strain, we propose that association of the C-terminal tail of the receptor with G(alpha) and Gbeta subunits is required for proper activation of the heterotrimeric G protein. Besides its described role in downregulation and in formation of preactivation complex, the results here shown indicate that the C-terminal tail of the receptor plays an active role in transmitting the stimulus of mating pheromone to the heterotrimeric G protein.
The molecular mechanism of constitutive activity of the G protein-coupled receptor for human parathyroid hormone (PTH1) has been examined by molecular dynamics (MD) simulations. The single point mutations H223R, T410P, and I458R, of the PTH1 receptor result in ligand-independent receptor activation. Extensive MD simulations indicate that each of the mutations, through different mechanisms, lead to very similar conformational changes of the third intracellular loop. The structural changes, centered on K405 in the C-terminus of the third intracellular loop, can be traced back to the single-point mutations by calculation of the forces and torques responsible for the collective motions of the receptor. This analysis indicates a direct correlation between the conformational preferences of the cytoplasmic loop and the mutations in different locations of the receptor: TM2 (H223R), TM6 (T410P), and TM7 (1458R). Given the pivotal role of the third intracellular loop of PTH1 in coupling to the G proteins, the structural changes induced by these single-point mutations may be responsible for the ligand-free activation of the receptor. These results coupled with the high-resolution structure of the third cytoplasmic loop of PTH1, previously determined in our laboratory, provide unique insight into the mechanism of ligand free activation of the PTH1 receptor.
Metabotropic glutamate (mglu) receptors are implicated in the regulation of many physiological and pathological processes in the CNS, including synaptic plasticity, learning and memory, motor coordination, pain transmission and neurodegeneration. Several recent studies have elucidated the molecular determinants of mglu receptor signaling and show that several mechanisms acting at different steps in signal propagation are involved. We attempt to offer an integrated view on how homologous and heterologous mechanisms regulate the initial steps of signal propagation, mainly at the level of mglu-receptor-G-protein coupling. Particular emphasis is placed on the role of phosphorylation mechanisms mediated by protein kinase C and G-protein-coupled receptor kinases, and on the emerging importance of some members of the regulators of G-protein signaling family, such as RGS2 and RGS4, which facilitate the GTPase activity that is intrinsic to the alpha-subunits of G(q) and G(i).