Distribution and ultrastructural localization of GEC1 in the rat CNS

We have previously demonstrated that GEC1 interacts with the kappa opioid receptor and GEC1 expression enhances cell surface expression of the receptor in Chinese hamster ovary cells. In this study, we generated an antiserum (PA629) directed against GEC1 in rabbits, characterized its specificity, and investigated distribution of GEC1 in tissues and in brain regions and spinal cord and its subcellular localization in hypothalamic neurons in the rat. Immunofluorescence staining demonstrated that PA629 recognized HA-GEC1 transfected into Chinese hamster ovary cells, but not HA-GABARAP or HA-GATE-16, although the three share high homology. Pre-incubation of PA629 with GST-GEC1, but not GST, abolished the staining. In immunoblotting, affinity-purified PA629 (PA629p) recognized GEC1, GABARAP and GATE-16. GEC1 migrated slower than GABARAP and GATE-16, with a M(r) of 16 kDa for GEC1 and M(r) of 14 kDa for GABARAP and GATE-16. Immunoblotting results showed that GEC1 level was higher in liver and brain than in lung and heart, and very low in kidney and skeletal muscle. GEC1 was present in all rat brain regions examined and spinal cord. Immunohistochemistry demonstrated that GEC1 immunoreactivity was distributed ubiquitously in the rat CNS with highly intense immunoreactivity in various brain nuclei and motor neurons of the spinal cord. Ultrastructural examination of neurons in the paraventricular nucleus of the hypothalamus showed that GEC1 was associated with endoplasmic reticulum and Golgi apparatus and distributed along plasma membranes and in cytosol. Coupled with our previous observation that GEC1 interacts with N-ethylmaleimide-sensitive factor, these findings strongly suggest that GEC1 functions in intracellular trafficking in the biosynthesis pathway and perhaps also the endocytic pathway. The widespread distribution of GEC1 suggests that GEC1 may be associated with many proteins, in addition to the kappa opioid receptor.