Rachel Sterne-Marr - US grants

9728179 Sterne-Marr G protein coupled receptors are membrane bound proteins which convert light and hormonal signals into intracellular messages. Uncoupling of the receptor from its immediate downstream signal transducer, the G protein, occurs rapidly following exposure to a stimulus and results in cessation of response to the hormone (desensitization). Receptor phosphorylation by the (adrenergic receptor kinase ( ARK) is hormone-dependent and promotes G protein/receptor uncoupling. Prior to hormone treatment ARK exists in an inactive state. Upon activation of the receptor by hormone, the catalytic activity of the kinase is stimulated and the receptor is phosphorylated, resulting in desensitization. Little is known about the structure of ARK, the molecular interactions which maintain the pre-active kinase in its basal state, and the conformation changes which convert the pre-active kinase into an active enzyme. The goals of this project are to understand how interactions within the ARK protein maintain this molecule in an inactive form and how interactions between ARK and a hormone stimulated receptor induce ARK activation. The techniques of mass spectrometry, chemical cross-linking and random targeted mutagenesis will be used to identify the amino acids in ARK which keep this protein in the pre-active form. Fusion proteins, site-directed mutagenesis, enzyme assays and ARK /receptor binding assays will be used to identify regions that interact with the receptors. Since ARK is a member of a family of G protein coupled receptor kinases which regulate G protein-coupled receptors, it provides a good model to study the structure and activation of this class of proteins. The interaction of a hormone or neurotransmitter with a specific receptor on the surface of a target cell initiates a chemical signaling event. A molecule known st a G protein, because it binds GTP, interacts with the receptor and downstream signaling molecules and a specific response occurs. To terminate the signal a specific protein, the adrenergic receptor kinase ( ARK) adds a phosphate group to the receptors. This research is directed at the mechanisms by which this protein becomes activated and interacts with the receptor. These studies will provide new information about activation of this class of proteins. ***

Individual cells of the body communicate with each other via hormones and neurotransmitters that are produced by one cell and then act on a target cell. The interaction of the hormone or neurotransmitter with a specific receptor sets off a signaling cascade within the cell. An essential mediator is a molecule called a G protein that interacts with both receptor and downstream signaling molecules leading to a response that is specific to the cell type. For example, when we are frightened, adrenalin is released into the bloodstream and it acts on target cells in the heart resulting in an increase in heart-rate. The appropriate intensity and duration of the signal are so important that multiple mechanisms have evolved which modulate such a signal. One mechanism of signal modulation involves hormone-induced inactivation of the receptor by a specific protein called G protein-coupled receptor kinase 2 (GRK2). Thus, GRK2 blocks the signal initiated by adrenalin and allows the cell to return to a basal state so that it can appropriately respond to the environment. Although GRK2 was first identified by its ability to modulate signaling by the adrenalin receptor, it is now known that GRK2 regulates many receptors, called G protein-coupled receptors, and these receptors in fact represent the largest class of proteins in the human genome. Furthermore, in addition to altering the receptor, GRK2 also binds the G protein. Thus, GRK2 "multi-tasks" to regulate hormone signaling. The goal of this project is to understand how GRK2 carries out hormone-stimulated functions such as inactivation of the receptor and interaction with the G protein. Since the shape of GRK2 is known in atomic detail, public databases and the literature can be used to predict, for example, how GRK2 interacts with receptors. The next step is to use molecular biological techniques to substitute individual amino acids to assess which portions of GRK2 are important for receptor interaction.
These studies will not only provide insight into the mechanism of receptor regulation by GRK2 but also define a mechanism of activation for this novel family of proteins. Defining receptor recognition sites will ultimately lead to the rational design of GRK2 inhibitors that may have therapeutic value. This project will be carried out at a predominantly undergraduate institution and will allow many students to participate in modern structural, molecular and cellular research. Extensive collaborations with investigators at research-intensive universities will ensure the infusion of the latest technology and this will be reflected in the cell biology, molecular biology and biochemistry curricula at this small liberal arts college. The principal investigator will maintain a strong record of engaging undergraduates in research (especially those from under-represented groups), and producing career scientists.

This project is supporting students in Biochemistry, Chemistry, Computer Science, and Physics for careers in biotechnology, nanotechnology, and information technology, which are important growth areas in the "Tech Valley," New York State's Capital Region. The program builds on existing experience with cohort programs and ongoing undergraduate-focused projects in nanotechnology curriculum development and research in chemistry, biochemistry, bioinformatics, physics, and computer science. Seminars on Tech Valley research and internship opportunities are regularly conducted, and mentoring throughout the student scholars' undergraduate careers is provided by an interdisciplinary cohort of faculty. The intellectual merit of the project lies in its focus on key interdisciplinary fields of inquiry which form some of the most rapidly developing and intellectually challenging areas of science. Furthermore, the project strengthens academic-industrial interactions that are situated in regional economic development. The broader impacts of the project are seen in its formal partnerships with two local community colleges to work with their students from the start to help prepare them for smooth transitions to the four-year science programs at the PI's institution. The student population at these "feeder" schools has a significant portion of minority and first-generation college students thus providing another aspect through which the project is able to exercise broad impact. Specific activities include mentoring and tutoring programs at the two-year college sites, joint science fairs, and participation by community college students in summer research opportunities. Finally, on-going relationships are in place with numerous local high-technology industries where graduating students have the opportunity to gain employment upon graduation.

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.