A recent paper by Chongguang Chen and his team in Philadelphia has identified the protein GEC1 as a microtubule associated protein (MAP) family member that interacts with the human kappa opioid receptor (hKOR). GEC1 interacts with the C-tail of the receptor (a G protein coupled receptor (GPCR)) and facilitates it's trafficking from the ER and Golgi to the cell surface.This localisation was demonstrated using two of Abcam's Subcellular Marker antibodies: Calreticulin antibody (ab4) and 58K Golgi protein antibody [58K-9] (ab6284).This is the first report into the biological function of GEC1 and the first demonstration that a GPCR interacts with a MAP family protein. It also gives significant information into the trafficking of the receptor in the biosynthesis pathway.
The C-tail of hKOR was used as bait in a yeast two-hybrid screening of a human brain cDNA library. GEC1 was one of the clones identified as
IHC - 58K Golgi protein antibody
Ab6284 positively staining formaldehyde HEK 293 at 1/200. Used in conjunction with goat anti mouse (Alexa 546)1/1500. Nuclear staining achieved with Hoechst.
interacting with hKOR, but was found to interact uniquely with the kappa form of the opioid receptor (OR), not with hMORs or hDORs. This interaction was confirmed in co-immunoprecipitation and pull-down experiments.
GEC1 itself was found to interact with Tubulin in pull-down studies. Similar to other MAP family members, it interacts with Tubulin via its N-terminal 22 amino acids.
Chen et al. went on to show that expression of GEC1 in cells with labelled hKOR increased the expression of hKOR by 90% and of cell-surface hKOR by 130%. This effect was not seen when a C-terminal mutant of hKOR was used. Furthermore, when an N-terminal truncated form of GEC1 was used (lacking its Tubulin binding domain) the effect was much reduced.
Immunoblotting and pulse-chase experiments demonstrated that GEC1 enhances the presence of 55 kDa fully glycosylated (mature) hKOR in plasma membranes and trans-Golgi.
GEC1 was found to colocalise with the ER and Golgi through immunohistochemical staining techniques using anti-Calreticulin and anti-58K Golgi protein antibodies from Abcam to mark the ER and Golgi respectively.
Together, this data suggests that GEC1 acts in the biosynthesis pathway of hKOR at the ER/Golgi to enhance the trafficking and maturation of hKOR. This leads to its increased cell surface expression and is dependent on GEC1's interaction with Tubulin and microtubules via its N-terminus. This interaction between a MAP protein and a GPCR is the first to have been demonstrated and may offer further insights into the trafficking and maturation of other GPCRs.
Reacts with Cow, Hamster, Human, Mouse and Rat.
Tested in Western blot, Immunoprecipitation, Immunocytochemistry, Electron Microscopy
Reacts with Rat, Human (see review), Dog and Hamster.
Tested in Western blot, Functional Studies, Immunofluorescence, Immunocytochemistry, Immunomicroscopy, Immunocytochemistry/Immunofluorescence