Effects of GPCR trafficking on the spatiotemporal control of signaling

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Project Summary/Abstract G protein coupled receptors (GPCRs) constitute a family of signaling receptors that regulate essentially every physiological process. Recent studies have begun to recognize the roles of G proteins and GPCRs in tumor biology. In particular, signaling by the β2-adrenergic receptor (β2-AR) and mutations in its cognate G protein, GNAS, have been associated with pancreatic ductal adenocarcinoma (PDAC) progression. Understanding the basic molecular mechanisms of GPCR signaling will be imperative for realizing their potential as novel therapeutic targets. Studies of ligand-dependent GPCR signaling have traditionally focused on receptor activation at the plasma membrane. However, the recent discovery that intracellular compartments also function as active signaling sites that elicit different signaling outcomes raises a critical biological question: how does GPCR trafficking dictate unique signals, and do these unique signals lead to unique cellular outcomes? The proposed work addresses this question by focusing on the role of endosomal GPCR activation in shaping cAMP- dependent transcriptional responses. First, starting in an established HEK293 cell model, the proposed studies will assess the mechanisms by which trafficking encodes the amount and timing of cAMP generation. Through manipulations of receptors and trafficking machinery, the relationship between endosomal residence time and cAMP signaling profiles will be tested to determine whether trafficking is sufficient to shape cAMP output or whether other factors, such as posttranslational modifications or interacting proteins, are required. Next, the proposed work will define how these distinct cAMP profiles are decoded to elicit a specific downstream response. Defined steps in the signaling cascade will be probed in order to ascertain whether the transcriptional response is sensitive to variations in cAMP generation or whether cells have evolved a uniform response to cAMP stimulus. Finally, the proposed studies will explore whether the principles delineated in the HEK293 system are relevant to our understanding of oncogenic signaling by β2-AR and GNAS in PDAC. The timing, location, and duration of cAMP generation in pancreatic ductal epithelial cells will be evaluated to determine if these cAMP signatures are regulated and consequential and how PDAC-associated mutations affect this relationship. These studies will advance an exciting area of fundamental cell biology—the spatiotemporal control of receptor-mediated signaling. The proposed research will also provide new insights into the mechanisms of oncogenic signaling by β2-AR and GNAS in PDAC and, more broadly, the paradigm of GPCR-induced cAMP signaling in cancer. This project will be carried out at the University of California, San Francisco under the mentorship of Dr. Mark von Zastrow, a leader in the field of GPCR trafficking and signaling, in collaboration with two experts in the fields of pancreatic cancer (Dr. Eric Collisson, UCSF) and oncogenic signaling by GPCRs (Dr. J. Silvio Gutkind, UCSD). In addition to the conceptual and experimental training from those labs, UCSF also offers a vibrant research community with expertise and facilities that will enable the successful realization of the proposed studies.