Elucidation of a eukaryotic chemorepulsion mechanism

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There is good evidence that some cells secrete chemorepellents that cause specific cell types to move away from them. However, much remains to be understood about the identity of the chemorepellents, their receptors, and the mechanisms they use to direct cell motility. We found that proliferating Dictyostelium cells secrete a protein called AprA, and that AprA is an extracellular signal that functions as a chemorepellent. Although AprA has little sequence similarity to mammalian proteins, AprA has predicted structural similarity to the human secreted dipeptidyl protease DPPIV, and shares functional properties with DPPIV. We found that human DPPIV is a chemorepellent for human and mouse neutrophils, and when applied locally, DPPIV can induce neutrophils to leave a tissue in two mouse models of a lung disease called acute respiratory distress syndrome (ARDS), and a mouse model of rheumatoid arthritis. To gain insights into a fundamental mechanism used in morphogenesis, ways to induce neutrophils to leave a tissue, and how one could augment or diminish the effect of a chemorepellent, we propose three specific aims to elucidate the molecular mechanisms used by AprA and DPPIV to cause chemorepulsion. Aim 1 is to identify the AprA receptor, since this plays a key role in the Dictyostelium chemorepulsion mechanism. Our preliminary work has identified a predicted G protein- coupled receptor called GrlH as a possible AprA receptor. We will carefully test this, and if GrlH is not the receptor, we will use several approaches to identify the receptor. Aim 2 is to elucidate the AprA chemorepulsion signal transduction pathway. Our preliminary data indicate that some components of the chemorepulsion mechanism are different from components used by the chemoattraction mechanism that allows Dictyostelium cells to aggregate toward cAMP. We will determine the extent to which the chemorepulsion mechanism uses known components of the chemoattraction mechanism, as well as use the power of unbiased genetic screens in Dictyostelium to identify additional components of the chemorepulsion mechanism. Aim 3 is to test the hypothesis that DPPIV uses a G protein-coupled receptor called PAR2 to induce neutrophil chemorepulsion, and use what we learn about the Dictyostelium chemorepulsion mechanism to determine the similarities and differences between the Dictyostelium and the human neutrophil chemorepulsion mechanisms. Together, this work combining molecular biology, genetics, cell biology, and biochemistry will help to elucidate eukaryotic chemorepulsion mechanisms, and identify potential drug targets that could enhance or inhibit chemorepulsion.