G Protein Coupled Receptors

Project Collaborators:

Dr. Jeffrey Becker University of Tennessee, Dr. Patricia Hinkle, Dr. Fred Naider College of Staten Island

Predicted transmembrane topology of C-terminally truncated Ste2p. The black circles
indicate residues that, when mutated, alter the FL1/FL2 ratio of fluorescence emission
of [Lys⁷(NBD),Nle¹²]α-factor bound to C-terminally truncated
Ste2p receptor. The gray circles indicate residues that, when mutated, alter the FL1/FL2
ratio of fluorescence emission of [DTyr³,Lys⁷(NBD),Nle¹²]α-factor
bound to Ste2p receptor. The cross-hatched residue (F55) alters fluorescence ratio of both
types of ligand. The arrows indicate the start- and end- points of the regions targeted
for mutagenesis in the four screened libraries.

The superfamily of G protein coupled receptors mediate cellular responses to a variety of sensory stimuli, hormones, growth factors, and neurotransmitters and are targets of a number of widely prescribed drugs. Signal transduction by these receptors occurs when an activated receptor interacts with a trimeric G protein in the cytoplasm, causing the G protein to release a bound molecule of GDP and to replace it with a molecule of GTP. The mechanisms by which ligand binding leads to activation of the intracellular G protein remain poorly understood. We are using a combination of genetic, biochemical, and biophysical methods to study the yeast α-factor receptor that prepares yeast cells for mating in response to binding of the mating pheromone, α-factor. Components of this yeast signal transduction pathway are homologous to, and in some cases functionally interchangeable with, corresponding components of mammalian signaling systems. However, the ability to easily select or screen for functional and non-functional receptors in yeast allows rapid identification of receptors with interesting mutations from among a randomly mutagenized population in a way that is not possible in mammalian systems. We are currently analyzing classes of mutations that can provide information on the structural changes associated with activation of receptors, on interactions between receptors, and different interactions with agonistic and antagonistic ligands that elicit different downstream signaling responses.

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