Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

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Transabdominal two-cavity approach for radical nephrectomy combined with inferior vena cava thrombectomy for malignant thrombus caused by renal cell carcinoma: a case series

Abstract Background Advanced renal cell carcinoma in some cases causes malignant intravascular thrombus with the potential for growth into the inferior vena cava or even the right atrium. Renal cell carcinoma is accompanied by malignant intravascular thrombus in up to 10% of cases. We present an overview of three patients diagnosed as having renal cell carcinoma with malignant intravascular thrombus requiring radical nephrectomy combined with inferior vena cava thrombectomy. Case presentation Three patients diagnosed as having renal cell carcinoma were indicated for renal cell carcinoma combined with inferior vena cava thrombectomy between 2014 and 2017 at our department: a 69-year-old white Caucasian woman, a 74-year-old white Caucasian woman, and a 58-year-old white Caucasian woman. According to the Novick classification of inferior vena cava tumor thrombus, there was one infrahepatic (level II) and two supradiaphragmatic (level IV) malignant intravascular thrombi. The average age of these patients was 67 years (range 58–74 years). All patients underwent radical nephrectomy combined with inferior vena cava thrombectomy through transabdominal approach. In patients with level IV malignant intravascular thrombus, transesophageal echocardiogram was used to guide the placement of the inferior vena cava cross-clamp above the diaphragm. In one patient the pericardium was opened to place a cross-clamp above a tumor just below the right atrium. There were no postoperative mortalities to date with an average follow-up of 23 months (range 2–48 months). To date, no patient has demonstrated recurrent inferior vena cava malignant intravascular thrombus requiring secondary inferior vena cava thrombectomy or any other treatment. A comparison of estimated blood loss and transfusion rate was not significantly different in all three cases. Conclusion Despite the technical complexity of the procedure, caval thrombectomy combined with radical nephrectomy currently represents the only radical treatment for renal cell carcinoma accompanied by malignant intravascular thrombus with good mid-term oncological outcomes