The cysteine-rich with EGF-Like domains 2 (CRELD2) protein interacts with the large cytoplasmic domain of human neuronal nicotinic acetylcholine receptor alpha 4 and beta 2 subunits

Using a yeast two-hybrid screening we report the isolation of a novel human protein, hCRELD2 beta , that interacts specifically with the large cytoplasmic regions of human nicotinic acetylcholine receptor (nAChR) alpha 4 and beta 2 subunits, both in yeast cells and in vitro. This interaction is not detected with nAChR alpha 7 and alpha 3 subunits. The hCRELD2 gene encodes for multiple transcripts, likely to produce multiple protein isoforms. A previously reported one has been renamed as CRELD2 alpha . Isoforms alpha and beta are expressed in all tissues examined and have the same N-terminal and central regions but alternative C-terminal regions. Both isoforms interact with the alpha 4 subunit. Within this subunit the interaction was localized to the N-terminal region of the large cytoplasmic loop. The CRELD2 beta protein is present at the endoplasmic reticulum where colocalized with alpha 4 beta 2 nAChRs upon cell transfection. Immunohistochemistry experiments demonstrated the presence of CRELD2 in the rat brain at sites where alpha 4 beta 2 receptors have been previously detected. Labeling was restricted to neuronal perikarya. Finally, CRELD2 decreases the functional expression and impairs membrane transport of alpha 4 beta 2 nAChRs in Xenopus leavis oocytes, without affecting alpha 3 beta 4 and alpha 7 nAChR expression. These results suggest that CRELD2 can act as a specific regulator of alpha 4 beta 2 nAChR expression.