D ifferences between isoforms

Genetic evidence has shown that each subtype is capable of mediating InsP^-induced Ca^"^ release from intracellular Ca^^ stores (Sugawara etal., 1997). However, the three subtypes differ in several ways, such as affinity for InsPj. For example, InsP^ interacts with the type n receptor with 3-fold higher affinity than the type I receptor (Newton et al., 1994; Sudhof

etal., 1991) and type III has been shown to have an approximately 10-fold lower affinity for InsPg than does type I (Newton et al., 1994). However, a more recent study has concluded that the affinity order is type I>type n>type III in both cell lines and rat tissues (Wojcikiewicz and Luo, 1998a). This data appears to be more reliable since it measured binding to whole InsPgR tetramers rather than to monomeric receptor fragments as in the earher studies. These latter differences in ligand-binding affinity appear to correlate well with the potency of InsPg as a Ca^^-mobilising agent (Wojcikiewicz and Luo, 1998a). The selective expression of a particular receptor type will thus influence the sensitivity of cellular Ca^^ stores to InsPg. The subtypes and splice variants are also differentially regulated (see below). The differences between the subtypes and splice variants may confer upon them distinct biological roles in the cells in which they are co-expressed.

1.5.6 InsPgR regulation

The large N-terminal cytoplasmic region provides the main region for cytoplasmic regulators to act. InsPgRs are regulated by small modulators such as InsPg and Ca^^ itself (discussed in more detail in section 1.5.7) (Taylor and Tray nor, 1995), adenine nucleotides (Bezprozvanny and Ehrlich, 1993; Missiaen etal., 1997), pH (Joseph et al., 1989), redox state (Bootman et al., 1992; Renard-Rooney et al., 1995) and phosphorylation (Joseph, 1996; Supattapone et al., 1988a; Wojcikiewicz and Luo, 1998b). For example, cAMP- dependent protein kinase (PKA) phosphorylates the InsPgR in intact cells and enhances InsPg-induced Ca^^ mobilisation in several cell lines (Wojcikiewicz and Luo, 1998b). However, PKA decreases sensitivity to InsPg in cerebellar microsomes (Cameron et al.,

1995; Supattapone et a l, 1988a), although this result may have been an artefact of the microsome preparation and may not apply to intact cerebellum cells.

InsPgRs are also modulated by larger protein-protein interactions with accessory proteins such as calmodulin (Patel etal., 1997; Yamada etal., 1995) and FKBP12 (Cameron et al.,

1995). The InsPgR has also been shown to play a role in the organisation of the intracellular Ca^"" stores. For example, in the Purkinje cells of the cerebellum the type I appears to participate in the formation of stacks of ER (Rusakov et al., 1993; Takei et al.,

1994) and in other tissues type I interacts with cytoskeletal proteins like ankyrin (Bourguignon and Jin, 1995; Bourguignon et al., 1993b; Joseph and Samanta, 1993) or actin (Rossier et al., 1991). More recently, the actin cytoskeleton has been shown to

mediate an interaction between the Sec6/8 complex (involved in exocytosis) and Ca^^ signaling complexes incorporating the type I and HI InsPgRs, in pancreatic acinar cells (Shin et a l, 2000). Furthermore, anti-Sec6 and anti-Sec8 antibodies inhibit Ca^^ signaling at a step upstream of Ca^"^ release by InsPg (Shin et a l, 2000). The InsPgR can thus integrate information from many signaling pathways to determine sensitivity to InsPg.

Subtypes and splice variants appear to be differentially regulated. For example, type HI InsPgRs, unlike type I InsPgRs, are not stimulated by sulfhydryl oxidation (by thimerosal) and are less sensitive to ATP as well as InsPg (Missiaen et al., 1998). Furthermore, type I InsPgR is much more susceptible to phosphorylation than types II and HI (Wojcikiewicz and Luo, 1998b). The neuronal (SII+) and non-neuronal (SII-) sphce variants of type I InsPgR (described above) also have different phosphorylation kinetics and patterns (Danoff

et at., 1991). With respect to CICR, Ca^^ differentially regulates the InsPg-affinity states of type I and type III InsPgRs (Cardy et al., 1997; Yoneshima et al., 1997) (see section 1.5.7). The subtypes also exhibit differences in their resistance to downregulation (Wojcikiewicz, 1995) (see section 1.5.9).

1.5.7 Regulation by Ca^^ and InsPg: regenerative Ca^^ transients

The InsPgRs of many tissues are biphasically regulated by cytosolic Ca^"^. During sustained exposure to Ca^^, responses to submaximal concentrations of InsPg are enhanced by modest increases in cytosolic Ca^"^ concentration (<300nM), while responses to most InsPg concentrations are inhibited by more substantial increases in Ca^^ concentration (>300nM) (Taylor, 1998). These effects were first described in guinea pig smooth muscle cells in 1990 (lino, 1990) when the terms CICR (Ca^^-induced Ca^^ release) and IICR (InsPg- induced Ca^^ release) were first used. It was also shown that InsPgRs need to be bound to InsPg in order to be activated by Ca^^ (Bezprozvanny et al., 1991; lino, 1990). It is thought that InsPg binding to a single receptor subunit (Yoshikawa et al., 1996) causes a large conformational change (Mignery and Sudhof, 1990) that exposes a Ca^^-binding stimulatory site (Marchant and Taylor, 1997; Parker et al., 1996). As the concentration of cytosolic Ca^^ increases, it can bind to a second inhibitory site which is accessible whether or not the receptor has InsPg bound (Taylor, 1998). The stimulation of InsPgRs by Ca^^ is thought to contribute to the regenerative recruitment of InsPgRs in intact cells, whereas the inhibitory effect of Ca^^ may provide the negative feedback that hmits the duration and amplitude of elementary Ca^^ release events (Berridge, 1997). More recently, this idea has been extended into a theory of lateral inhibition. This hypothesises that InsPg switches its receptor from a state in which only an inhibitory Ca^^-binding site is accessible to one in which only a stimulatory site is available. This ensures that Ca^^ released by an active InsPgR may rapidly inhibit its unliganded neighbours, but it cannot terminate the activity of a receptor with InsPg bound, thus providing increased sensitivity to InsPg and allowing rapid graded recruitment of InsPgRs (Adkins and Taylor, 1999).

The biphasic effect of cytosolic has been observed in tissues expressing predominantly the type I, II or III InsP^R (Swatton et al., 1999; Taylor, 1998) and is thus widely thought to be a feature of aU subtypes. However, another study on single-channel function of receptor subtypes suggests that they may differ in this form of regulation. Whereas single type I channel activity is a biphasic function of Ca^^ concentration, single type n channel activity is not inhibited by high Ca^^ concentration (Ramos-Franco et al.,

1998). Finally, one way in which Ca^"^ regulates InsP^-induced Ca^^ release is by regulating InsP^ binding. For example, Ca^^ has different effects on InsPg binding to types I and III InsPgR (Cardy etal., 1997; Yoneshima et al., 1997). One study (on recombinant mammalian InsPgRs in insect Sf9 cells) found that Ca^^ increases inhibit InsPg binding to type I receptors by causing 50% to adopt a conformation with undetectable affinity for InsPg. In contrast, the same Ca^^ increases unmask InsPg-binding sites on type HI receptors but, as Ca^^ increases further, there is a substantial decrease in their affinity for InsPg (Cardy et al., 1997). In other words, it appears under these conditions that Ca^^ causes only inhibition of binding to type I whereas it biphasically regulates InsPg binding to type

in

InsPgRs. Accessory proteins and the direct effect of Ca^^ on channel opening also play a role in determining the biphasic regulation of InsPg-induced Ca^^ release.

The experiments used in describing biphasic regulation were mostly not done in intact cells but on permeabilised cells or single InsPgR channels incorporated into planar lipid bilayers (Bezprozvanny et al., 1991; Swatton et al., 1999). In a physiological situation, this form of regulation may be significantly affected by InsPgR density since it is dependent upon diffusion of Ca^^ from the locality of one receptor to that of another. Downregulation of InsPgR expression (see below) will thus have consequences for CICR through the InsPgR. The underlying mechanisms of CICR are not completely understood. However, as suggested above, it is generally proposed that the two effects of Ca^^ are probably mediated by two distinct Ca^^-binding sites, which may reside on the InsPgR itself or on accessory proteins (Taylor, 1998).

In addition to regulation by binding to Ca^^, InsPgRs are regulated in a complex manner by InsPg itself (Marchant and Taylor, 1998). Firstly, InsPgRs exhibit cooperative responses to InsPg, enabling an all-or-nothing response (Callamaras et al., 1998; Marchant and Taylor, 1997; Meyer etal., 1988) and thus preventing spontaneous activation. Binding of InsPg to its receptor unmasks a site to which Ca^"^ can then bind, and when all four subunits of the receptor have bound both ligands, the intrinsic Ca^^ channel opens to its maximal activity (Marchant and Taylor, 1997). Limiting spontaneous opening of channels is important since such positive feedback (CICR) could potentially lead to the generation of inappropriate Ca^^ transients or empty all Ca^^ stores and raise cytosolic Ca^^ to toxic levels (Dowd, 1995).

Secondly, experiments suggest that the InsPgR may be inactivated by InsPg (Hajnoczky and Thomas, 1994; Hajnoczky and Thomas, 1997; Marchant and Taylor, 1998). However, these experiments were carried out in permeabilised hepatocytes and the role of calcium in InsPg-induced receptor inactivation remains controversial. Also, hepatocytes have predominantly type II InsPgRs (De Smedt e ta l, 1997) so this type of inactivation may not be a feature of all subtypes. In these experiments, InsPg-induced receptor activation was followed by an obligatory time-dependent inactivation, which appeared to be a direct consequence of InsPg binding (Hajnoczky and Thomas, 1994; Hajnoczky and Thomas,

1997). As described above, binding of InsPg to each of its four receptor subunits unmasks a site to which must bind before the channel can open to maximal activity. However, a different study in support of the above experiments suggests that InsPg binding also initiates a slower switch of the receptor to a higher-affinity but less active (more closed) conformation which is about 40% less able to mediate Ca^^ release (Marchant and Taylor,

1998). Thus it appears that InsPgRs may be switched to a less active conformation/state by both Ca^^ and InsPg. These two pathways must together determine the permeability of the InsPgR to Ca^^ and thus the local rise in cytosolic Ca^^ concentration.

In addition to InsPg-binding cooperativity and Ca^"^- and InsPg-regulated channel activation and partial inactivation, there are other elements which play a part in generating repetitive Ca^^ transients (Meyer and Stryer, 1988). InsPg is continuously destroyed by a phosphatase and therefore new InsPg must be generated. This may involve the stimulation of a Ca^^-dependent PLC by released Ca^^ (positive feedback). Furthermore, as well as being pumped out of the cell, Ca^^ ions that enter the cytosol are continuously pumped back into the ER by Ca^^-ATPase pumps. When the Ca^^ stores have been refilled to a critical level, the next spike may be generated. It has been proposed that the time required for the Ca^"^ level in the ER to reach a threshold sets the interspike interval (Meyer and Stryer,

1988).

In document The role of inositol 1,4,5-triphosphate receptors in mammalian oocytes and preimplantation embryos (Page 32-35)