REFERENTES TEÓRICOS

The catalytic subunit of P P l, PPlc exists as various isoforms (a, p, y l, y2 and Ô, Cohen, 1988, Dombradi et al, 1990, Sasaki et al, 1990), the a, P, y l and y2 isoforms have all been detected in brain, where they show distinct subcellular localisation: P P lp localizes to a discrete area of the soma, while P P ly l is highly enriched in dendritic spines and presynaptic terminals of cultured neurons (Strack et al, 1999). This subunit forms a 1:1 complex with a regulatory subunit (Hubbard and Cohen, 1993) of which several types exist, targeting the enzyme to specific subcellular locations. The glycogen-bound regulatory subunit of PPl is termed the G-subunit, forming the holoenzyme PPl G (Strâlfors et al, 1985) while muscle contains PPIM (Alessi etal, 1992), the nucleus PPIN (Beullens et al, 1992,1993) and the cytosol PPIS, which has 1-2 as its regulatory subunit (DePaoli-Roach etal, 1994).

PPlc is regulated by binding to its various regulatory/targeting subunits and to inhibitor proteins such as I-l, 1-2 and the brain form of I-l, DARPP-32 (Hemmings et al, 1984a). The best-characterized targeting protein is the glycogen-binding or G subunit, which confers association of PPl with skeletal muscle glycogen. P Pl G dephosphorylates the enzymes of glycogen metabolism. It is active when the two subunits are bound together; this binding is regulated by phosphorylation of G at two sites. Phosphorylation of site I (serine 48) is required for the association of G and P P lc (Hubbard and Cohen, 1989), while site 2 (serine 67) phosphorylation promotes dissociation of the free catalytic subunit

into the cytosol, greatly reducing its ability to dephosphorylate glycogen phosphorylase , and glycogen synthase (Hubbard and Cohen, 1989, Dent et al, 1990). The activating effect of site 1 phosphorylation is overridden by phosphorylation of site 2. Application of adrenaline to skeletal muscle cells causes a rise in intracellular cAMP via the action of adenylate cyclase, leading to activation of PKA and phosphorylation of the G subunit of PPIG at site 2 resulting in its dissociation and inactivation (Dent et al, 1990).

and inhibits PPlc. Thus hormone-induced dissociation of PPIG is coordinated with I-l activation to inhibit protein dephosphorylation in mammalian skeletal muscle. I-l- phosphate is dephosphorylated by PP2B, causing it to dissociate from PPlc, while site 2 phosphorylated by PKA on the G subunit is dephosphorylated by PP2A, PP2B and PP2C

in vitro (Hubbard and Cohen, 1989).

PPl is widely distributed in neurons, being present in membrane fractions (Sim et al,

1994), dendritic spines (Guimet et al, 1995), the postsynaptic density (Strack et al,

1997a), synaptic junctions (Shields et al, 1985), and associated with neurofilaments (Strack et al, 1997b). PPlc also associates with brain microtubules. A PPl targeting subunit recently identified is the microtubule-associated protein tau; P Pl and possibly other PPs are involved in regulating microtubule stability (Liao et al, 1998). A recently identified neuronal PPl binding protein, spinophilin, localises PPl to dendritic spines in the vicinity of PPl targets such as AMPA-type glutamate channels (Allen et al, 1997, Guimet et al, 1995). It has recently been discovered that P P lc binds to the PKA anchoring protein AKAP220, thus co-localising with its opposing enzyme PKA in the vicinity of shared substrates (Schillace and Scott, 1999).

PPl is involved in diverse cellular processes including glycogen metabolism, calcium transport, muscle contraction, protein synthesis and intracellular transport (Cohen, 1989, Shenoliker and Naim, 1990) and also appears to have an important role in the regulation of mitosis (Doonan and Morris, 1989, Axton et al, 1990). In the brain, as in other tissues, P Pl is mainly associated with the particulate fraction (Cohen and Cohen, 1989, Sim et al, 1994), although in contrast to other tissues this membrane-bound PPl appears to have a low specific activity (Sim et al, 1994). Functions of PPl in brain appear to include regulation of the neuronal cytoskeleton by dephosphorylation of microtubule-associated and neurofilament proteins, and regulation of CaM kinase II, which is instrumental in gene expression, neurotransmitter synthesis and release, postsynaptic responses and cytoskeletal rearrangements (Strack et al, 1997a, Shields et al, 1985, Kennedy, 1998, Braun and Schulman, 1995). The synaptic localization of P P ly l indicates that this

isoform is involved in the regulation of synaptic phosphoproteins such as neurotransmitter receptors and ion channels implicated in synaptic plasticity (Strack et al, 1999). The three inhibitor proteins of PPl, I-l, 1-2 and DARPP-32 are present in brain (Macdougall et al,

1989, Hemmings et al, 1992, Oui met et al, 1984).

PPl has a well-characterised role in regulation of dopaminergic signalling cascades in which its effects are opposed by DARPP-32. DARPP-32 is found at high concentrations in certain brain regions and is absent from virtually all other tissues (Hemmings and Greengard, 1986). The distribution of DARPP-32 and I-l is often complementary, suggesting some difference in function (Hemmings et al, 1992, Alder and Barbas, 1995). The main site of DARPP-32 expression is in the basal ganglia, which contains neurons having dopamine Dj receptors coupled to adenylate cyclase (Hemmings and Greengard,

1986). It is also found in some glial cells, including astrocytes, which are known to contain dopamine-sensitive adenylate cyclase (Walaas and Greengard, 1984, Guimet et al, 1984) and in which the neuronal form of I-l could not be detected (Gustafson et al,

1991).

Dopamine, acting on D^ receptors causes activation of PKA which phosphorylates DARPP-32 on threonine-34 (Hemmings et al, 1984b). In its phosphorylated form, DARPP-32 inhibits PPl, which controls the phosphorylation state and consequently the physiological activity of various neuronal phosphoproteins such as neurotransmitter receptors, ion channels, ion pumps and transcription factors (Shenoliker and Naim, 1991). In astrocytes, the role of PPl also includes the dephosphorylation of most sites on GFAP, an intermediate filament protein which is restricted mainly to astrocytes and undergoes multisite phosphorylation (Vinadé and Rodnight, 1996). The role of DARPP-32 in dopamine neurotransmission was studied using mice that lack the protein (Fienberg et al,

1998). Fienberg and co-workers found that mutation of the DARPP-32 gene markedly reduced, and in some cases abolished, various responses to dopaminergic agonists and antagonists. The authors conclude that a cascade involving dopamine receptor-mediated activation of DARPP-32, inhibition of P P l, and increased phosphorylation of neuronal

substances is an obligatory component in dopaminergic neurotransmission. Since disturbances in dopaminergic neurotransmission have been implicated in several major neurological and psychiatric disorders including Parkinsonism, drug addiction and schizophrenia, drugs that mimic or block the inhibitory effects of DARPP-32 on PPl might prove useful pharmacological agents (Fienberg et al, 1998).