Expression of VACM-1 Protein in Cultured Rat Adrenal Endothelial Cells is Linked to the Cell Cycle

Abstract

The vasopressin-activated calcium-mobilizing (VACM-1) protein is a unique arginine vasopressin (AVP) receptor which shares sequence homology with the cullins, genes involved in the regulation of cell cycle transitions. Unlike either cullins or AVP receptors, however, VACM-1 is expressed exclusively in the vascular endothelial cells and in the renal collecting tubule cells. In order to test the hypothesis that the expression of VACM-1 might be correlated with the cell cycle, and to establish an endothelial cell model for the VACM-1 receptor, we examined VACM-1 expression in rat adrenal medulla endothelial cells (RAMEC). Northern and Western blot analyses of mRNA and protein from RAMEC identified presence of 6.4 kb mRNA and a Mr 81 kDa protein, respectively. Immunostaining of RAMEC with anti-VACM-1 antibodies and Western blot analyses indicated that in RAMEC, VACM-1 protein expression is dependent on the cell cycle. VACM-1 protein virtually disappears during the S phase and localizes to the cytosol during cell division and to the cell membrane at the completion of cytokinesis. Furthermore, pretreatment of RAMEC with anti-VACM-1 specific antibodies increased basal levels of Ca2+and attenuated the AVP-dependent increase in cytosolic Ca2+. In summary, these results indicate that VACM-1 protein expression in RAMEC membrane is linked to the cell cycle, and consequently, VACM-1 may be involved in the regulation of cell division.

Untitled

“…With these techniques, we demonstrated a major CUL-5 transcript of ~7.4 kb and a major immunoreactive protein of M r ~ 82 kDa in primary HMECs, immortalized but non-tumorigenic MCF-10A breast epithelial cells, ER-α positive MCF-7 breast cancer cells, and ER-α negative MDA-MB-231 breast cancer cells, which is consistent with previous reports [7,17-19]. While CUL-5 expression was unchanged in breast cancer cells versus breast epithelial cells, the cells used for these studies came from unsynchronized subconfluent proliferating cultures, and cell cycle dependent expression of some CUL family members has been described [19,20]. Investigation of cell cycle dependent expression of CUL-5 is crucial to gain insight into both the mechanism(s) regulating the expression and potential cellular function(s) of CUL-5.…”

SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function

“…Accompanying these deficiencies, SENP8 knockout cells also showed aberrant G1/S cell cycle progression and accelerated cell growth (Figure 6). Interestingly, these cell cycle alterations partially phenocopy previously reported defects associated with the loss of either Cul1 (Dealy et al, 1999; Wang et al, 1999; Chen and Li, 2010) or Cul5 function (Burnatowska-Hledin et al, 2001; Buchwalter et al, 2008; Bradley et al, 2010; Ma et al, 2013a; Willis et al, 2017). Through an unbiased proteomic screen in which we enriched for K-ε-GG remnant-containing peptides in MG132-treated cells, we were able to identify several candidate substrates with deregulated ubiquitylation status and stability in the SENP8 knockout cells that could potentially contribute to G1/S progression defects.…”

Hypertension in mice with transgenic activation of the brain renin-angiotensin system is vasopressin dependent

“…There is some evidence for expression of V 2 receptors in extrarenal tissues such as lung (16) and cerebellum (33), though their physiological significance in these tissues is unclear. Finally, AVP is also an agonist at the VACM-1 receptor, also known as Cullin-5, where it elicits calcium mobilization in endothelial cells and renal collecting ducts (5,6). Our determination that mesenteric artery V 1A receptors were downregulated in sRA mice but renal V 2 receptor expression was unchanged may suggest a greater role for AVP-mediated renal water retention in the hypertension of sRA mice.…”