Mutations in polycystin-1 (PC1) lead to autosomal-dominant polycystic kidney disease (ADPKD), a leading cause of renal failure for which no treatment is available. PC1 is an integral membrane protein, which has been implicated in the regulation of multiple signaling pathways including the JAK/STAT pathway. Here we show that membrane-anchored PC1 activates STAT3 in a JAK2-dependent manner, leading to tyrosine phosphorylation and transcriptional activity. The C-terminal cytoplasmic tail of PC1 can undergo proteolytic cleavage and nuclear translocation. Tail-cleavage abolishes the ability of PC1 to directly activate STAT3 but the cleaved PC1 tail now coactivates STAT3 in a mechanism requiring STAT phosphorylation by cytokines or growth factors. This leads to an exaggerated cytokine response. Hence, PC1 can regulate STAT activity by a dual mechanism. In ADPKD kidneys PC1 tail fragments are overexpressed, including a unique ∼15-kDa fragment (P15). STAT3 is strongly activated in cyst-lining epithelial cells in human ADPKD, and orthologous and nonorthologous polycystic mouse models. STAT3 is also activated in developing, postnatal kidneys but inactivated in adult kidneys. These results indicate that STAT3 signaling is regulated by PC1 and is a driving factor for renal epithelial proliferation during normal renal development and during cyst growth.

The effect of homocysteine on alpha‐2‐macroglobulin (a2M) function and structure was investigated by mass spectrometry, circular dichroism, isoelectric focusing, surface plasmon resonance, and UV‐vis spectroscopy. Structural analysis of homocysteinylated‐a2M revealed substantial conformational changes in native a2M compared to the untreated a2M. Proteinase‐activated a2M was more resistant towards homocysteinylation than its native form. Proteinase binding function of a2M was shown to be decreased in the presence of L‐homocysteine but not L‐cysteine, which may indicate that high levels of homocysteine in inflammatory states may decrease the ability of a2M to clear proteinases. Mass spectrometry analysis of homocysteinylated‐a2M identified 8 cysteine‐containing peptides suggesting that at the time of alkylation these cysteine residues were in the reduced form. Moreover, a new disulfide link was observed between C255‐C264, indicating that the free cysteine residues that appear upon homocysteinylation tend to reform disulfide bridges and therefore lead to “scrambled” disulfide bonds that can have a profound effect not just on the three‐dimensional structure of the a2M tetramer but also on its functions. This work was supported by a grant (HL52234, DWJ) from the Heart, Lung, and Blood Institute of the National Institutes of Health.

Alpha-2-macroglobulin (a2M), a 720-kDa plasma protein, functions as an extracellular proteinase inhibitor and carrier of cytokines, growth factors and hormones. Disulfide bonds are susceptible for thiol-exchange reactions with homocysteine (Hcy). The structure of a2M contains 96 cysteine residues and one can assume that some of these disulfide bridges might interact with Hcy. Using 35S-D,L-homocysteine, we show that a2M is homocysteinylated in both its native and in the protein-proteinase complex (“activated” a2M). The stoichiometry of Hcy binding to native and activated a2M is dose-dependent: treatment of native and activated a2M with 25 and 50 μM 35S-D,L-Hcy at 37°C for 4 h yielded approximate Hcy:a2M ratios of 2:1 and 4:1 for native a2M, and 3:1 and 6:1 for activated a2M. Trypsin-protein esterase activity of a2M was shown to be decreased by 12–35% in homocysteinylated-a2M compared to untreated a2M, suggesting that high levels of Hcy in the blood may affect the proteinase-binding function of a2M. This might explain why proteolytic activity remains high in inflammatory tissue fluids and why cytokines and growth factors that usually bind to a2M continue to play important roles in the progression of inflammation and atherosclerosis.