Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

The endoplasmic reticulum chaperone gp96 is required
for the cell
surface expression of a narrow range of proteins, including toll-like
receptors (TLRs) and integrins. To identify a more comprehensive repertoire
of proteins whose cell surface expression is dependent on gp96, we
developed plasma membrane profiling (PMP), a technique that combines
SILAC labeling with selective cell surface aminooxy-biotinylation.
This approach allowed us to compare the relative abundance of plasma
membrane (PM) proteins on gp96-deficient versus gp96-reconstituted
murine pre-B cells. Analysis of unfractionated tryptic peptides initially
identified 113 PM proteins, which extended to 706 PM proteins using
peptide prefractionation. We confirmed a requirement for gp96 in the
cell surface expression of certain TLRs and integrins and found a
marked decrease in cell surface expression of four members of the
extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence
of gp96. Other novel gp96 client proteins included CD180/Ly86, important
in the B-cell response to lipopolysaccharide. We highlight common
structural motifs in these client proteins that may be recognized
by gp96, including the beta-propeller and leucine-rich repeat. This
study therefore identifies the extended LDL receptor family as an
important new family of proteins whose cell surface expression is
regulated by gp96.