Grant: Discovery Grants Program - Individual

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological functions, such as smell, taste, vision, secretion, neurotransmission, metabolism, inflammatory and immune response. GPCR signaling is thought to stem from the cell surface where receptor interactions with external stimuli can be transduced into cellular responses. Accumulating evidence indicates that many GPCRs also signal from inside the cell. Peroxisomes participate in central pathways of cellular metabolism such as ß-oxydation of fatty acids and detoxification of reactive oxygen species. Peroxisomes have complex biogenesis and degradation pathways, are actively distributed intracellularly and possess their own targeting and translocation machineries. They are dynamic structures whose morphology, abundance, content and function can be rapidly regulated in response to diverse signals. Peroxisomes have been overlooked so far in GPCR biology. Through our research program on the proteins that interact with and regulate TPß (thromboxane A2 receptor), we identified several peroxisomal proteins involved in: A) targeting and translocation of proteins to peroxisomes, B) motility/distribution of peroxisomes, and C) various peroxisomal metabolic pathways. Of particular interest in the latter group is ACOX1, the enzyme involved in the first step of fatty acid ß-oxydation, producing H2O2. TPß co-localization with peroxisomal markers was detected by immunofluorescence microscopy. Interaction between the receptor and peroxisomal proteins were confirmed by co-immunoprecipitation and western blot experiments. To the best of our knowledge, this is the first evidence of the presence of GPCRs in peroxisomes. We will concentrate on the 3 following short-term objectives for the next 5 years: 1)TPß localization to peroxisomes will be studied by electron microscopy at the endogenous level in HeLa cells. Radioligand binding experiments will also be carried out on purified peroxisomes from HeLa cells to further study the presence of TPß. The orientation of TPß in the peroxisomal membrane will be determined. The molecular mechanisms involved in targeting TPß to peroxisomes will be studied. 2)The effects of the stimulation, the blockade and the presence of TPß in peroxisomes on the number, motility, and distribution of this organelle will be characterized. The molecular mechanisms involved in these processes will be explored.  3)The interaction of TPß and peroxisomal enzymes will be studied. We will first focus on the interaction with ACOX1. We will determine whether ACOX1 regulates the functional responses of TPß. We will address whether TPß modulates the activity of ACOX1. To the best of our knowledge, GPCRs have never been described in peroxisomes and their function in this organelle is still unknown. Furthermore, our program will provide significant advancement in our understanding of the molecular regulation of peroxisome biology in response to cellular signals.