Functional properties of leptin receptor isoforms internalization and degradation of leptin and ligand-induced receptor downregulation

Long (ObRb) and short (ObRa) leptin receptor isoforms are thought to play e ssential roles in mediating leptin signaling and the transport and degradat ion of leptin, respectively. Although the capacity of these cloned receptor species to mediate signal transduction has been reported, there is no info rmation on the ability of individual receptor species to mediate leptin int ernalization and degradation or to undergo ligand-induced downregulation. W e therefore studied these parameters in Chinese hamster ovary (CHO) cells s tably expressing either ObRa or ObRb isoforms of the leptin receptor. We de termined that both ObRa and ObRb mediated internalization of I-125-labeled leptin by a temperature- and coated pit-dependent mechanism. Both ObRa and ObRb also mediated degradation of I-125-leptin by a lysosomal mechanism, an d this was more efficiently mediated by ObRa in these cells. Neither leptin internalization nor degradation by ObRa was affected by mutation of the co nserved Box 1 motif. By studying deletion mutants of ObRa, we found that ef ficient internalization was dependent on a motif located between amino acid s 8 and 29 of the intracellular domain of ObRa. Exposure of cells expressin g ObRa or ObRb to unlabeled leptin for 90 min at 37 degrees C produced down regulation of available surface receptors, and this effect was of greater m agnitude in cells expressing ObRb. Whereas CHO cells expressing the growth hormone receptor showed marked downregulation of ligand binding after expos ure to dexamethasone (DEX) or phorbol myristic acid (PMA), PMA had no effec t on expression of ObRa or ObRb, and DEX reduced binding to cells expressin g ObRb by 15%. Thus, the two leptin receptor isoforms, ObRa and ObRb, media te leptin internalization by a coated pit-dependent mechanism, leptin degra dation by a lysosomal pathway, and ligand-induced receptor downregulation. The differential capacity of the two receptor isoforms may relate to the di fferent roles of the receptor isoforms in the biology of leptin.