Biochemical and Genetic Studies of the Gαo Signaling Mechanism

Gα_o is by far the most abundant Gα protein in the brain, and every neurotransmitter has receptors that activate this G protein. Gα_o was discovered over 35 years ago, yet we still do not know what “effector” proteins this G protein directly binds and regulates to carry out its functions. We do know from C. elegans genetics that Gα_o directly opposes Gα_q signaling, and ultimately Gα_o signaling inactivates neurons, preventing them from carrying out neurotransmitter release.

We have recently carried out rapid, gentle immunopurifications of Gα_o protein complexes from both mouse brain and C. elegans lysates, and identified the proteins in these complexes by mass spectrometry. We found a number of proteins that copurify with Gα_o in both mouse and worms, and verified the association of Gα_o with these proteins by co-immunoprecipitation/Western blots. Intriguingly, several of these proteins are regulators of Gα_q signaling and/or neurotransmitter release, exactly the types of proteins that could plausibly mediate Gα_o signaling. We are carrying out further biochemical studies of the association of Gα_o with these proteins, analyzing how Gα_o may regulate activity of these proteins, and are using C. elegans genetics to analyze the functional significance of these proteins in Gα_o signaling.

Organization of G protein coupled receptors in neural circuits

C. elegans has 27 G protein coupled neurotransmitter receptors and about 150 G protein coupled neuropeptide receptors, which together facilitate signaling among the 302 neurons (of 118 types) found in the worm. Interestingly, humans have about the same number of G protein coupled neurotransmitter and neuropeptide receptors, even though we have ~80-100 billion neurons. In C. elegans, if each receptor is expressed in 20-30 types of neurons (a reasonable estimate based on existing data), that means the average neuron expresses about 6 neurotransmitter and 32 neuropeptide receptors. As one of the first steps to understanding how a neural circuit functions, we need to know which neurons in the circuit express which receptors.

We are undertaking a large-scale project to map which cells in C. elegans express each of its 27 G protein coupled neurotransmitter receptors. For every receptor, we have created fosmid-based GFP reporter transgenes that cause all cells expressing that receptor to fluoresce green. We are currently mapping out which cells express each receptor within the egg-laying circuit, and then we will broaden our analysis to map neurotransmitter receptor expression in the entire nervous system. We have also begun work mapping the cellular expression patterns of the neuropeptide receptors. The neural signaling maps we produce will be essential tools for future work analyzing any neural circuit or behavior in C. elegans.

An egg-laying circuit as a model for understanding neural circuits in general

Much of what is known about neural circuits comes from past studies of very simple circuits in crustaceans, in which their large neurons could be impaled with electrodes to study their activity. Unfortunately, modern genetic methods cannot be applied to crustaceans, limiting what we can learn from them. Conversely, the nematode C. elegans is readily amenable to genetic manipulation via mutations and transgenes and has one of the best-characterized nervous systems of any organism.