Our group is interested in how cells process information, particularly through heterotrimeric G proteins. We study the molecular mechanisms that these proteins use to detect, sort, amplify and convey information; and how these mechanisms are regulated to provide G protein signaling modules with adaptability and diversity.
G protein signaling modules detect a vast number of wildly diverse incoming signals: hormones, neurotransmitters, growth and differentiation factors, odors, tastes, chemoattractants, nutrients, pheromones and light. They are the targets of a majority of all prescription drugs, and are involved in the etiology of numerous diseases. Their sensitivities to stimuli range at least from 10-10 M through 10-2 M, their response times vary from <0.01 seconds to >100 seconds, and their ability to amplify signals can exceed 1000-fold.
Despite their striking capabilities, G protein modules consist of only three or four components -- a receptor, a G protein, an effector protein and (sometimes) a GTPase-activating protein (GAP). Further, these proteins use a single conserved biochemical mechanism for signaling.
G protein modules are a fascinating example of how a relatively small group of densely interacting regulatory proteins can form a highly complex signaling module with broadly adaptable behaviors. Their many physiological and pharmacological roles give their mechanism added practical importance.
The GTPase Cycle — how G proteins work
G proteins convey information by traversing a tightly regulated cycle of GTP binding and hydrolysis. G proteins are activated by binding GTP on their Gα subunits, such that both their Gα and Gβγ subunits can regulate specific effector proteins. The G protein remains activated until it hydrolyzes bound GTP to GDP, which does not activate. Signal amplitude is simply the balance of the rates of activation (GDP release and GTP binding) and deactivation (GTP hydrolysis). The intrinsic rates of GDP/GTP exchange and of GTP hydrolysis are slow. Exchange is catalyzed by the receptor, and hydrolysis is accelerated by the GAP.
Mammals express about 1000 G protein-coupled receptors including receptors for hormones and neurotransmitters, odors and pheromones and diverse other regulatory compounds, as well as the photoreceptor rhodopsin. Plants and fungi add to this diversity. G protein-coupled receptors (GPCRs) are all integral membrane proteins with similar overall structures. Mammals express about 20 G proteins, defined by their Gα subunits, that interact selectively with subsets of these receptors and in turn activate subsets of effector proteins. G proteins are peripheral membrane proteins, and are found primarily on the inner face of the plasma membrane. G protein-regulated effectors are diverse and unrelated in structure. More than 20 are now known, but new effectors continue to be discovered.
What are the questions?
While basic G protein biochemistry is fairly well understood, how these reactions combine to form a signal-processing network poses interesting and important questions.
Why are some GAPs inhibitors and some GAPs accelerators?
By accelerating GTP hydrolysis, a GAP can inhibit signaling, and some GAPs are primarily inhibitors. Others, however, do not inhibit and instead accelerate signal turn-off when stimulus is removed. We are studying how GAPs can change signaling speed or signal amplitude independently. Control of this decision involves the stability of receptor binding to the G protein, the ability of the Gβγ subunits to inhibit GAP activity, and precise control of the rates of protein-protein interactions during each round of the GTPase cycle. These ideas are discussed in more depth in a recent essay in Current Biology (2008).
How can a G protein-regulated effector also act as a GAP?
The effector phospholipase C-β (PLC-β) is stimulated by the Gq family of G proteins, but PLC-β is also an active Gq GAP. PLC-ß1 increases the rate of hydrolysis of GTP bound to Gq about 1200-fold. How can PLC-β (and other effector GAPs) respond to activated Gq without suppressing Gq activation? What is the mechanism whereby PLC-β acts as a GAP, and how is that activity controlled? We are measuring PLC-β activation using fluorescent biosensors to monitor activation and deactivation in real time along with the binding of PLC-β to Gq.
What is the sequence of binding interactions among receptor, Gα and Gβγ, GAP and effector during the GTPase cycle?
The rates and duration of protein-protein binding is central to the control of G protein signaling. We are using fluorescently labeled proteins to measure these interactions in real time with millisecond resolution.
Computational analysis of these simultaneous reactions is necessary for their understanding.
These reactions, simple individually, combine to form a complex, non-linear signal transduction module. We are using computational analysis of the multiple simultaneous events of the GTPase cycle to determine how they coordinately regulate activation and deactivation of signaling. Assembling the models and determining what reactions can be included based on the availability of data are both challenging. Computational modeling allows us to compare observed signaling behaviors with those predicted numerically and thus ask if the proposed reaction system accurately describes the signaling process. Because of the complex simultaneous reactions among the involved proteins, only such analysis can accurately compare mechanism and observation.
How do multiple signals sum?
G protein modules branch. A single receptor can stimulate several different G proteins, a G protein can listen to many different receptors and regulate several different effector proteins, and an effector can listen to several different G proteins. In a cell’s G protein network, incoming signals can sum to produce additive outputs, either positively or negatively (one signal may inhibit another). Alternatively, interactions can be less than or greater than additive: two incoming signals may be necessary to produce a significant cellular response. We are studying how G protein signaling through phospholipase C-β may be simply additive or highly synergistic depending on which G protein subunits regulate which isoform of the enzyme. Strong synergism between two inputs can create a coincidence detector, or Boolean AND gate, such that the cell responds only when both stimuli occur simultaneously.
How does Gz modulate signaling
despite its slow rate of intrinsic GTP hydrolysis?
Gz, a member of the Gi family of G proteins that is expressed in neurons and some neuroendocrine cells, is unusual in that it hydrolyzes bound GTP extremely slowly, with a deactivation lifetime of about 7 minutes at 30°C. Several Gz -selective GAPs increase this rate over 600-fold. We are trying to determine what molecular interactions allow this effect, and how the slow responses of Gz contribute to neuronal function. We are also studying how Gz GAP activity is regulated in neurons.
Our work is supported by the National Institute of General Medical Sciences, the Welch Foundation and the Greer Garson and E.E. Fogelson Distinguished Chair in Medical Research.