Activation of intracellular signal transduction cascades frequently involves increased phosphoinositide hydrolysis following stimulation of phospholipase C. Inositol 1,4,5-trisphosphate (IP 3 ), 1 a second messenger produced by phosphoinositide hydrolysis, mediates Ca 2ϩ release from intracellular stores by binding to IP 3 -sensitive Ca 2ϩ channels, thereby increasing their "open" probability (1). IP 3 receptors derive from at least three different genes, constituting types I, II, and III, which are approximately 70% identical at the amino acid level but differ in distribution and regulation (reviewed in Refs. 1-3). An assembly of four 260-kDa subunits forms the receptor. Each subunit consists of a cytoplasmic, amino-terminal IP 3 binding domain, a coupling domain, and a Ca 2ϩ channel pore of six transmembrane segments (2-4). Type I is further diversified by alternative RNA splicing, resulting in two main forms, of which the longest (SIIϩ, containing the 40 amino acid residues 1693-1732) is specifically expressed in neurons (2). One or more IP 3 receptor forms have been found in virtually all cell types examined (reviewed in Ref. 2), but particularly high amounts of type I IP 3 receptor are seen in smooth muscle cells and in cerebellar Purkinje neurons. Calcium release mediated by IP 3 receptors appears to be an essential step for the induction of long term depression (LTD) in Purkinje cells (5).A number of different mechanisms modulates IP 3 receptor function, including binding of ATP, fatty acids, and calcium (reviewed in Ref. 2); a number of neurodegenerative processes (6, 7); and phosphorylation of the IP 3 receptor by specific protein kinases. cAMP-dependent protein kinase (PKA) phosphorylates the type I IP 3 receptor both in vitro and in vivo (8 -11) and has also been reported to phosphorylate type II and III in intact cells (12). Ca 2ϩ /calmodulin-dependent protein kinase II, protein kinase C and the tyrosine kinase Fyn have also been reported to phosphorylate the type I IP 3 receptor (13)(14)(15)(16)(17). In addition, the receptor may undergo autophosphorylation (18). Early work indicating that the neuronal IP 3 receptor (SIIϩ) can be phosphorylated by cGMP-dependent protein kinase (PKG) (8) was later confirmed by in vitro experiments (19 -21). Likewise, the nonneuronal type I IP 3 receptor (SIIϪ) found in smooth muscle cells, also termed the G 0 protein (22, 23), is a substrate for phosphorylation by both PKA (10) and PKG (19,20,(23)(24)(25). Recent reports of IP 3 receptor phosphorylation by PKA and PKG in hepatocytes (26 -28), kidney cells (11), and platelets (29, 30) support these observations. Phosphorylation of the IP 3 receptor by PKA and PKG represents a possible mechanism for cross-talk whereby cyclic nucleotides can modulate IP 3 -mediated regulation of Ca 2ϩ levels (20, 31). Because cAMP and cGMP levels in most cells are regulated by various extracellular signals, identification of phosphorylation sites labeled by these kinases is of interest. Amino acid sequencing indicated that PKA phosphor...

Ca"-induced degradation of the neuronal inositol(l,4,5)-trisphosphate receptor, a protein which regulates Ca"-release from intracellular stores, has been examined. The IP,-receptor, immunopurified from rat cerebellum, appeared to be an excellent substrate for purified Ca'+-activated neutral protease (calpain). Incubation of membranes or immunopurified IP,-receptor with Ca" and cerebellar cytosol also resulted in degradation of the receptor. Two main fragments with approximate molecular masses of 130 and 95 kDa were generated, both of which appeared to derive from the carboxyterminal Ca"-channel-contaming part of the protem. These data suggest that activation of the IP,-receptor, by causing increases in intracellular [Ca"], might result in degradation of the N-terminal, IP,-binding part of the receptor.

The type I inositol 1,4,5-trisphosphate (IP(3)) receptor is selectively down-regulated in several neurodegenerative diseases, including Alzheimer's disease, Huntington's chorea, and ischemia, all conditions in which apoptotic neuronal loss occurs. In the present study, we used a neuronal cell line, human neuroblastoma SH-SY5Y cells, to investigate whether the levels of IP(3) receptor are changed during apoptosis in these cells. Following induction of apoptosis by staurosporine, the immunoreactivity of the type I IP(3) receptor in microsome preparations from SH-SY5Y cells was reduced within 2 h, with a further reduction during subsequent hours. Immunoblot analyses, using antibodies to poly(ADP-ribose) polymerase and spectrin breakdown products, revealed proteolysis of these caspase-3 substrates within 3 h, confirming that IP(3) receptor cleavage is an early consequence of apoptosis. In vitro incubation of SH-SY5Y microsomes or immunopurified IP(3) receptor from rat cerebellum with recombinant caspase-3 led to generation of immunoreactive breakdown products similar to those observed in intact cells, suggesting that the type I IP(3) receptor is a potential substrate for caspase-3. Preincubation of the neuroblastoma cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-fluoromethyl ketone prevented IP(3) receptor degradation. These results show that the type I IP(3) receptor is a substrate for caspase-3 in neuronal cells and indicate that apoptotic down-regulation of IP(3) receptor levels may contribute to the pathology of neurodegenerative conditions.

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