Convergent trafficking pattern of leptin after endocytosis mediated by ObRa-ObRd.

The cellular effects of leptin are dependent on the receptor subtypes that mediate the signaling and fate of endocytosed leptin inside the cells. In this study, we examined the differences in receptor expression, endocytosis, intracellular degradation, and exocytosis of a trace amount of leptin in cells overexpressing ObRb and short forms of the leptin receptor. The relative contribution of proteasomes and lysosomes in the intracellular fate of leptin was also determined. There were three unusual findings: (1) all receptor subtypes could mediate the binding and endocytosis of leptin, although ObRb was expressed at a lower level than ObRa, ObRc, and ObRd after transient transfection. This indicates that ObRb can be a transporting receptor. (2) Once internalized, the intracellular degradation pattern and exocytosis of leptin were independent of the receptor subtype. (3) Endocytosed leptin could remain intact for at least 1 h. This stability was further enhanced by inhibition of lysosomal activity. Thus, the intracellular pool of intact leptin may allow prolonged biological functions for this adipokine.

Identification of the TBX5 transactivating domain and the nuclear localization signal.

TBX5 is a member of the T-box gene family and encodes a transcription factor involved in cardiac and limb development. Mutations of TBX5 cause Holt-Oram syndrome (HOS), an autosomal-dominant condition with congenital cardiac defects and forelimb anomalies. Here, we used a GAL4-TBX5 fusion protein in a modified yeast-one hybrid system to elucidate the TBX5 transactivating domain. Using a series of deletion mutations of TBX5, we narrowed down its functional domain to amino acids 339-379 of its C-terminal half; point mutagenesis analysis then showed that the loss of amino acids 349-351 abolished transactivation. This result was confirmed in mammalian cells. Furthermore, wild-type TBX5, but not TBX5 with mutations at the amino acids 349-351, has ability to inhibit NCI-H1299 cell growth also suggesting that these amino acids are crucial for the TBX5 function in mammalian cells. In addition, to identify the nuclear localization signal of TBX5, we searched for cluster of basic amino acids. We found that the deletion of the KRK sequence at amino acids 325-327 mislocalizes TBX5 to cytoplasm, suggesting that these amino acids serve as a nuclear localization signal. These studies enhance our understanding of the structure-function relationship of TBX5 and suggest that truncation mutations of TBX5 could cause HOS through the loss of its transactivating domain and/or the nuclear localization signal.