Signaling between cells is required to regulate higher order functions in the body such as heart rate, glucose metabolism, and sensory perception. Signaling molecules such as hormones and neurotransmitters interact with receptors on target cells and initiate a cascade of events that culminates in a physiological effect. One type of receptor, termed G protein-coupled receptor (GPCR), transduces many intercellular messages and represents a large class of proteins encoded by the human genome. While hormones and neurotransmitters initiate signaling cascades, intracellular proteins are required to terminate and thereby regulate receptor signaling. The focus of this study is a regulatory protein called GPCR kinase (GRK2) that plays a crucial role in interacting with activated receptors to terminate the signal. One goal of this investigation is to map the binding interface between GRK2 and the receptors it regulates. It is hoped that these studies will illuminate the mechanism by which this novel class of proteins is activated and ultimately lead to the rational design of GRK2 inhibitors. This project will be carried out at a predominantly undergraduate institution and will expose a wider population of future scientists to structural, molecular and cellular techniques. Extensive collaborations with investigators at research-intensive universities will ensure the infusion of the latest technology and this will be reflected in the genetics, cell biology, molecular biology and biochemistry curricula. The principle investigator will maintain strong record of engaging undergraduates, especially those from under-represented groups, in research and producing career scientists. To further broaden the participation of under-represented populations, the PI will continue to work with Siena College's Higher Education Opportunity Program (HEOP), a New York State college program for economically and academically disadvantaged students, to recruit HEOP students to participate in the proposed research.
Regulation of Cellular Signaling: Mechanism of Activation of G Protein-Coupled Receptor Kinase 2 (GRK2) Individual cells of living organisms respond to cues generated in their environment and by other cells. The interaction of light, hormones, neurotransmitters, and many other molecules with a specific receptor on the surface of the target cell initiates signaling event that leads to a cell-specific response. In most cases, it is only appropriate for cell signaling to occur for a short period of time. By analogy to a computer monitor, the information needs to be "refreshed" to be of value. The visual system is "refreshed" on a millisecond timescale to allow organisms to respond to their environment, while hormonal signaling is updated within seconds to minutes of the original stimulus. These "refreshing" events serve to modulate the duration and amplitude of the signaling. Receptors that respond to light and adrenaline are members of a very large class of receptors called G protein-coupled receptor or GPCRs. The 2012 Nobel Prize in Chemistry that was awarded to Robert J. Lefkowitz and Brian K. Kobilka spotlighted the importance of these receptors. Because the duration of signaling is important, cells utilize multiple strategies to achieve termination of the signal. One ubiquitous mechanism of signal termination involves a signal-induced receptor modification, called phosphorylation, to make the receptor less responsive to the stimulus. Phosphorylation is a decoration that introduces a localized concentration of negative charges to the receptor and thereby changes its iteracting partners and therefore function. Kinases are enzymes (protein catalysts) that carry out phosphorylation; GPCR kinases or GRKs are kinases that phosphorylate GPCRs but only when the receptor has responded to an external cue. A regulatory loop is thereby created: the receptor responds to a signaling cue but also activates the GRK to turn off the receptor and "refresh" the signaling system. The goal of this project was to determine what parts (amino acids) of GRK2 are required for interaction with the GPCR (binding) and what parts are required to convert the enzyme from an inactive to an active form (activation). The approach was to introduce mutations in the GRK2 DNA that subtly change the surface properties of GRK2. The design of these "mutations" required models of GRK2 predicted from crystal structures of GRK2 and related kinases. To test the role of these mutations, we used in vitro and intact cell assays of receptor binding and phosphorylation. We found that one region of GRK2, the amino terminal alpha helix, uses one of its surfaces to directly interact with GPCRs (pink). The opposite face of this helix interacts with a region known as the active site tether (both cyan). These interactions stabilize the GPCR docking site. These atoms shown in cyan play two functions: they seem to be necessary 1) to allow the amino terminal region to interact with GPCRs and 2) for activation of the kinase (converting it from the inactive to the catalytically active form). One amino acid, shown in blue, seems to be required for enzyme activation but not for receptor binding. Thus, the GPCR interaction surface of GRK2 is similar to that suggested for other GRKs. However, different GRK family members (there are 7) also make unique interactions with GPCRs. Since there are hundreds of GPCRs in mammalian genomes, we can begin to dissect the role of GRKs in regulating these important receptors. Another goal of the project was to prepare career scientists. During this granting period, 26 undergraduate students and one high-school student were trained in the lab. Fifteen of them are currently pursuing careers as researchers in the molecular biosciences with others pursuing careers in the health professions. Ten of the trainees were women and five were from groups underrepresented in science. Four of the trainees are currently performing graduate or postdoctoral work at Stanford University, Yale University and Cornell University. One of these students obtained her doctorate in 3½ years. Thirteen students published their undergraduate work in Biochemistry, Methods in Enzymology and the Journal of Biological Chemistry. Through awards and other opportunities, the PI was able to reach out to the Siena College Board of Trustees, the President of the College, graduates and their guests. Finally, in collaboration with others on campus, the PI played an instrumental role in 1) securing an NSF STEM award, and 2) institutionalizing undergraduate research through the creation of the Siena College Center for Undergraduate Research and Creative Activity.
