Generation of human soluble leptin receptor by proteolytic cleavage of membrane-anchored receptors

The leptin receptor (ObR) exists in multiple isoforms. In rodents, a solubl e isoform is generated by alternative splicing; but in humans, there is no mRNA encoding soluble receptor (leptin binding protein). We investigated th e hypothesis that human leptin binding protein can be generated by proteoly tic cleavage of membrane-anchored leptin receptors (ObRb and ObRa). Leptin binding protein of similar size to that previously detected in human serum was detected by HPLC in medium of cells transfected with ObRa. ObRa exhibit ed higher expression at the cell surface than ObRb and generated greater le vels of leptin binding protein. Ligand-mediated immunofunctional and immuno fluorometric assays revealed that the leptin binding protein in medium boun d both leptin and an ObR-specific antibody and that the level of leptin bin ding protein correlated with receptor expression at the cell surface. Phorb ol 12-myristate-13-acetate and N-ethylmaleimide increased the accumulation of leptin binding protein, an indication that the production of leptin bind ing protein was up-regulated by PKC and sulfhydryl group activation. The pr otease inhibitors, TNF alpha protease inhibitor I and Immunex compound 2, c ould inhibit the production of leptin binding protein, indicating that the enzyme responsible for leptin binding protein cleavage belongs to the metal loprotease family. In conclusion, human leptin binding protein is generated by proteolytic cleavage of membrane-anchored leptin receptor by a metallop rotease.