The IP3R (inositol 1,4,5-trisphosphate receptor) Ca2+-release channel is known to be sensitive to thiol redox state. The present study was undertaken to characterize the number and location of reactive thiol groups in the type-I IP3R. Using the fluorescent thiol-reactive compound monobromobimane we found that approx. 70% of the 60 cysteine residues in the type-I IP3R are maintained in the reduced state. The accessibility of these residues was assessed by covalently tagging the IP3R in membranes with a 5 kDa or 20 kDa MPEG [methoxypoly(ethylene glycol) maleimide]. MPEG reaction caused a shift in the mobility of IP3R on SDS/PAGE that was blocked by pretreatment of the membranes with dithiothreitol, N-ethylmaleimide, mersalyl or thimerosal, indicating that MPEG reactivity was specific to thiol groups on the IP3R. Trypsin cleavage of the type-I IP3R generates five defined domains. In cerebellum membranes, MPEG reacted over a 5 min interval with tryptic fragment I and fragment III, but not fragments II, IV or V. Fragment I appears as a doublet in cerebellum membranes, corresponding to the presence and absence of the SI splice site in this region (SI is a spliced domain corresponding to amino acids 318-332). Only the fragment I band corresponding to the SI(+) splice form shifted after reaction with MPEG. Expression of SI(+) and SI(-) spliced forms in COS cell microsomes confirmed this result. The MPEG-induced shift was not prevented when the cysteine residue present in the SI splice domain (C326A) or the remaining seven cysteine residues in fragment I were individually mutated. Of the combination mutations screened, only the mutation of C206/214/326A blocked MPEG reactivity in fragment I. We conclude that a set of highly reactive cysteine residues in fragment I are differentially accessible in the SI(+) and SI(-) splice variants of the type-I IP3R.
2؉dependence and structural requirements of the IP 3 R proteasomal degradation pathway.The activation of inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 2 by IP 3 initiates Ca 2ϩ mobilization from the ER and triggers the Ca 2ϩ signal that underlies alterations in cell function elicited by a diverse array of cell surface stimuli (1, 2). A commonly observed characteristic of cells is that they adapt their responses when chronically stimulated. In the case of cell surface receptors, this is usually the result of phosphorylation and/or internalization. Wojcikiewicz et al. (3,4) were the first to note that chronic stimulation of cultured SHSY-5Y neuroblastoma cells with carbachol for 6 h causes 90% loss of type I IP 3 R protein by a mechanism involving the marked acceleration of IP 3 R degradation. Subsequently, similar effects on IP 3 R degradation have been described in many different experimental systems with many different agonists (5-16). Downregulation of IP 3 R protein is associated with a decrease in the ability of IP 3 to mobilize Ca 2ϩ (4,8). More recently it has been shown that the frequency of elementary Ca 2ϩ puffs that trigger propagating Ca 2ϩ waves in cells is decreased by chronic agonist stimulation (14). The physiological significance of IP 3 R down-regulation is unknown. However, down-regulation of receptors would be expected to inhibit the global Ca 2ϩ signal elicited by all Ca 2ϩ mobilizing agonists (heterologous desensitization). If this is the only component of the signal transduction system that is down-regulated, then an imbalance in signaling pathways would exist and may have profound consequences for cellular responses. A physiologically relevant example is the response of pituitary cells to GnRH receptor agonists (17). Mammalian GnRH receptors lack a C-terminal tail and therefore do not bind arrestin. Consequently these receptors desensitize and internalize only very slowly. However, sustained activation of GnRH receptors does lead to desensitization of gonadotrophin hormone secretion, which suggests the presence of adaptive mechanisms distal to the cell surface receptor. Treatment with GnRH receptor agonists produce a rapid and pronounced degradation of IP 3 Rs, and this is associated with a marked reduction in the IP 3 -mediated Ca 2ϩ signal (15, 18). The mechanism of IP 3 R degradation has not been established. Previous studies have shown that chronic elevation of IP 3 and IP 3 binding to the receptor are required to facilitate IP 3 R degradation (19,20). It has been proposed that the sustained elevation of IP 3 causes the IP 3 R to adopt a conformation that exposes sites that become ubiquitinated. The proteasome pathway then degrades the ubiquitinated IP 3 receptor. The regions of the IP 3 R involved in ubiquitination have not been determined. Pretreatment of cells with the SERCA pump inhibitor thapsigargin has been found to inhibit agonist-mediated IP 3 R degradation, suggesting that Ca 2ϩ also plays a role in IP 3 R degradation (4). In the present study we have examined IP 3 R degra...
Within 5 years, 18 % of our study population developed colorectal cancer. Most of these malignancies were found within the 50-80-year age group and located predominantly in the right colon and distally in the sigmoid and rectum. While distal cancers may be visualized by flexible sigmoidoscopy, those located more proximally may be missed, necessitating the need for a full colonoscopy. Although staging was similar between the two groups, Group 1 tumors were less aggressive despite having appeared within 5 years. As a result of our incidence of colorectal cancer within a 5-year interval, a shorter period for routine colonoscopy may be considered.
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