Location of gp330/α₂-m receptor-associated protein (α₂-MRAP) and its binding sites in kidney: Distribution of endogenous α₂-MRAP is modified by tissue processing

The α₂-macroglobulin receptor-associated protein (α₂-MRAP) is a 39 to 44 kDa protein that copurifies with the α₂-macroglobulin receptor (α₂-MR/LRP) and also with gp330, a highly glycosylated protein located within kidney proximal tubules and glomerular podocytes. Both gp330 and the α₂ -macroglobulin receptor are members of the low density lipoprotein receptor family but the physiological ligands for gp330 are unknown. In order to understand potential functions of the α₂-MRAP, specific anti-α₂-MRAP antibodies were used for immunocytochemical studies on paraformaldehyde lysine periodate (PLP)-fixed rat kidneys and on snap-frozen/acetone-fixed tissue. Conflicting results were obtained. After PLP fixation, α₂-MRAP was detected almost exclusively in rough endoplasmic reticulum (RER) cisternae; cell surface staining as virtually absent. In snap-frozen tissue, intense staining of the proximal tubule brush border was found, with little or no cytoplasmic staining. A series of experiments showed that during incubation of snap-frozen tissues, endogenous α₂-MRAP is released in soluble form from its intracellular location (i.e., the RER) and binds to gp330 on the brush border of proximal tubules. The location of binding sites for α₂-MRAP in rat kidney was also examined, using an (α₂-MRAP-IgG fusion protein. In both snap-frozen and PLP-fixed tissues, this probe bound exclusively to brush borders, and not to intracellular sites. Our results demonstrate: a) that in renal proximal tubule cells, (a₂-MRAP is located predominantly in the RER, b) that α₂-MRAP-binding sites are present on gp330, which is on the proximal tubule brush border, and c) that the apparent brush border localization of α₂-MRAP detected in snap-frozen sections is due to an artifactual redistribution of endogenous α₂-MRAP that occurs during tissue processing.