Location of gp330/alpha 2-m receptor-associated protein (alpha 2-MRAP) and its binding sites in kidney: distribution...

The alpha 2-macroglobulin receptor-associated protein (alpha 2-MRAP) is a 39 to 44 kDa protein that copurifies with the alpha 2-macroglobulin receptor (alpha 2-MR/LRP) and also with gp330, a highly glycosylated protein located within kidney proximal tubules and glomerular podocytes. Both gp330 and the alpha 2-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 alpha 2-MRAP, specific anti-alpha 2-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, alpha 2-MRAP was detected almost exclusively in rough endoplasmic reticulum (RER) cisternae; cell surface staining was 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 alpha 2-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 alpha 2-MRAP in rat kidney was also examined, using an alpha 2-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, alpha 2-MRAP is located predominantly in the RER, b) that alpha 2-MRAP-binding sites are present on gp330, which is on the proximal tubule brush border, and c) that the apparent brush border localization of alpha 2-MRAP detected in snap-frozen sections is due to an artifactual redistribution of endogenous alpha 2-MRAP that occurs during tissue processing.