ERD2 PROTEINS MEDIATE ER RETENTION OF THE HNEL SIGNAL OF LRPS RECEPTOR-ASSOCIATED PROTEIN (RAP)

The 39 kDa receptor-associated protein (RAP) is a receptor antagonist that interacts with several members of the low density lipoprotein (LD L) receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors, Our recent studies have d emonstrated that RAP is an endoplasmic reticulum (ER) resident protein and an intracellular chaperone for the LDL receptor-related protein ( LRP), The HNEL sequence at the carboxyl terminus of RAP represents a n ovel ER retention signal that shares homology with the well-characteri zed KDEL signal, In the present study, using immunoelectron microscopy we demonstrate that cells stably transfected with human growth hormon e (GH) tagged with either KDEL (GH+KDEL) or HNEL (GH+HNEL) signals exh ibit ER and cis-Golgi localization typical of ER-retained proteins, Ov erexpression of not only GH+HNEL but also GH+KDEL cDNA in transfected cells results in saturation of ER retention receptors and secretion of endogenous RAP indicating that the two signals interact with the same ER retention receptor(s). The role of RAP in the maturation of LRP is further supported by the observation that functional LRP is reduced a bout 60% as a result of decreased intracellular RAP. Pulse-chase label ing and immunolocalization studies of ERD2.1 and ERD2.2 proteins in tr ansfected cells demonstrate a long half-life and Golgi localization fo r both receptors. Finally, overexpression of either ERD2.1 or ERD2.2 p roteins significantly increases the capacity of cells to retain both K DEL and HNEL-containing proteins. Taken together, our results thus dem onstrate that ERD2 proteins are capable of retaining the novel ER rete ntion signal associated with RAP.