Stimulation of cell surface receptors results in the formation of the second messenger myo-inositol 1,4,5-trisphosphate (IP 3 ) 1 via the activation of phospholipase C (1). IP 3 mobilizes intracellular calcium by binding to a family of receptors (IP 3 Rs) that act as ligand-gated calcium channels (2, 3). IP 3 Rs are tetramers, and full-length sequences of at least three different isoforms have been identified by molecular cloning (reviewed in Ref. 4). Both homo-and heterotetramers are found in cells expressing more than one isoform (5-8).The acute regulation of IP 3 Rs occurs primarily through feedback effects of cytosolic Ca 2ϩ and/or by phosphorylation of the receptor (reviewed in Refs. 4 and 9). However, prolonged exposure of cells to agonists has also been shown to alter the expression of IP 3 R protein levels. For example, a decreased expression of IP 3 R isoforms occurs in response to chronic stimulation of muscarinic receptors in SH-SY5Y human neuroblastoma and cerebellum granule cells (10, 11), and cholecystokinin and bombesin receptors in AR4-2J pancreotoma cells (12). Down-regulation of IP 3 Rs has also been observed in Xenopus oocytes having a sustained activation of phospholipase C as a result of expressing a constitutively active mutant of G␣ q (13). Based on metabolic labeling studies, it has been concluded that the decreased expression of IP 3 Rs caused by carbachol treatment of SHY-SY5Y cells is due to enhanced degradation of IP 3 R protein rather than an inhibition of receptor biosynthesis (10). Down-regulation of IP 3 Rs has been suggested to be a possible mechanism of heterologous desensitization of responses to Ca 2ϩ -mobilizing hormones (12, 13). Examples of increased expression of IP 3 R protein have also been reported. The type I IP 3 R isoform was elevated in human promyelocytic leukemic HL-60 cells treated with retinoic acid and 1,25-dihydroxyvitamin D 3 (14, 15). This effect was shown to be due to enhanced transcription of the type I IP 3 R gene (14,15). Recently, large increases in the expression of the type III isoform were reported to accompany apoptosis induced in B and T lymphocytes (16).To further study the mechanisms regulating biosynthesis and degradation of the IP 3 R protein, we have examined the effect of chronic agonist stimulation of WB cells. This nontransformed, cultured rat liver epithelial cell line (17) was used as an experimental model because these cells contain high levels of both type I and type III IP 3 R isoforms (6,18). In the present study we report that both isoforms are down-regulated when WB cells are chronically stimulated by Ang II. We have characterized this response and present evidence to suggest that the Ang II-mediated degradation of IP 3 Rs involves activation of a ubiquitin-proteasome pathway.

COS-7 cells were transiently transfected with type I and type III myo-inositol 1,4,5-trisphosphate receptor (IP 3 R) isoforms to study the processes underlying assembly and oligomerization of these tetrameric proteins. A FLAG epitope was engineered on to the N terminus of the type III IP 3 R to distinguish the transfected from the endogenous isoform. This was not necessary for the type I IP 3 R, since the endogenous levels of this isoform were extremely low. Based on sucrose gradient analysis, the transfected type I or FLAG-type III IP 3 Rs assembled into tetramers. Confocal immunofluorescence experiments confirmed that the constructs were primarily targeted to the endoplasmic reticulum. Recombinant type I IP 3 R expressed in COS cells over a 48-h period showed a negligible capacity to form hetero-oligomers with endogenous type III IP 3 Rs, based upon coimmunoprecipitation assays. However, substantial formation of hetero-oligomers was observed between recombinant receptors when the cells were simultaneously transfected with type I and FLAG-type III IP 3 Rs. Co-immunoprecipitation experiments using lysates from metabolically labeled cells allowed the quantitation of homo-and hetero-oligomers in cells transfected with different ratios of type I and FLAG-type III IP 3 R DNA. These studies show that the relative expression level of the two isoforms influences the fraction of hetero-oligomers formed. However, the proportion of hetero-oligomers formed were less than predicted by a binomial model in which the association of subunits is assumed to be random. In doubly transfected cells, the early kinetics of 35 S label incorporation into homotetramers showed a lag period corresponding to the time taken to synthesize a full-length receptor. However, hetero-oligomers were synthesized with a longer lag period, suggesting that there may be kinetic constraints that favor homo-oligomers over hetero-oligomers.

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