Sub-cellular localization of insulin-regulated membrane aminopeptidase, IRAP to vesicles in neurons Ruani N. Fernando,* Susan E. Luff, Anthony L. Albiston* and Siew Yeen Chai* *Howard Florey Institute, The University of Melbourne, Parkville, Victoria, Australia  Monash Micro Imaging, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia Abstract Angiotensin IV and LVV-hemorphin 7 promote robust enhan- cing effects on learning and memory. These peptides are also competitive inhibitors of the insulin-regulated membrane aminopeptidase, suggesting that the biological actions of these peptides may result from inhibition of IRAP activity. However, the normal function of IRAP in the brain is yet to be determined. The present study investigated the sub-cellular distribution of IRAP in four neuronal cell lines and in the mouse brain. Using sub-cellular fractionation, IRAP was found to be enriched in low density microsomes, while lower levels of IRAP were also present in high density microsomes, plasma membrane and mitochondrial fractions. Dual-label immuno- histochemistry confirmed the presence of IRAP in vesicles co- localized with the vesicular maker VAMP2, in the trans Golgi network co-localized with TGN 38 and in endosomes co- localized with EEA1. Finally using electron microscopy, IRAP specific immunoreactivity was predominantly associated with large 100–200 nm vesicles in hippocampal neurons. The location, appearance and size of these vesicles are consistent with neurosecretory vesicles. IRAP precipitate was also detected in intracellular structures including the rough endo- plasmic reticulum, Golgi stack and mitochondrial membranes. The sub-cellular localization of IRAP in neurons demonstrated in the present study bears striking parallels with distribution of IRAP in insulin responsive cells, where the enzyme plays a role in insulin-regulated glucose uptake. Therefore, we pro- pose that the function of IRAP in neurons may be similar to that in insulin responsive cells. Keywords: AT 4 receptor, angiotensin IV, GLUT4, P-LAP, oxytocinase. J. Neurochem. (2007) 102, 967–976. The insulin-regulated membrane aminopeptidase (IRAP, EC 3.4.11.3) is a transmembrane aminopeptidase and a marker of specialized vesicles containing the insulin responsive trans- porter, GLUT4 (Keller et al. 1995). In insulin responsive tissue, including adipose and skeletal muscle, IRAP traffics with GLUT4 within intracellular compartments and translo- cates with GLUT4 to the plasma membrane in response to specific stimuli, including insulin (Slot et al. 1991a; Good- year et al. 1992; Chen et al. 1997; Ross et al. 1997). In the brain IRAP is expressed specifically in neurons, with high levels of expression in the pyramidal cell layer of CA1 to CA3, of the hippocampus (Albiston et al. 2001), throughout the cortex (including in the entorhinal, retrosplenial, parietal, insular, frontal and prefrontal cortices), nuclei of the amyg- dala, medial septum, hypothalamus and in motor neurons (Fernando et al. 2005). In addition, IRAP and GLUT4 are co- localized in the same cells in selected regions of the brain, including the pyramidal cells in CA1–CA3 of the hippocam- pus and spinal cord motor neurons in the ventral horn (Fernando et al. 2005). The role of IRAP in the brain is yet to be fully elucidated, although the memory-enhancing effects of the peptides, angiotensin IV (Ang IV) and LVV-hemorphin 7 (LVV-H7), which are also competitive inhibitors of the enzyme, are well established (Albiston et al. 2003). Central administration of Ang IV or LVV-H7, facilitates spatial learning and conditioned-avoidance memory in normal rats Received January 15, 2007; revised manuscript received February 2, 2007; accepted: February 28, 2007. Address correspondence and reprint requests to Siew Yeen Chai, Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: sychai@hfi.unimelb.edu.au Abbreviations used: DAB, 3,3¢-diaminobenzidine; dbcAMP, dibutyryl cyclic adenosine monophosphate; ECL, electrogenerated chemilumi- nescence; FBS, fetal bovine serum; HDM, high density microsomes; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LDM, low density microsomes; M/N, mitochondria and nuclei; NHS, normal horse serum; PBS, phosphate buffered saline; PM, plasma membrane; SBH, sucrose-buffered HEPES; TBS-T, Tris-buffered saline plus Tween; TGN, trans-Golgi network. Journal of Neurochemistry , 2007, 102, 967–976 doi:10.1111/j.1471-4159.2007.04659.x Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 967–976 967