Tissue Distribution and Subcellular Localization

Differences in tissue distribution provide a mechanism for diversity in the physiological functions of PLCs, some of which have been confirmed by transgenic studies in knockout mice (Reviewed in section 4). Of the PLCβ isozymes, β1 and β3 are the most widely expressed, at particularly high levels in specific regions of the brain197, 212-216, with

appears to be expressed primarily in hematopoeitic cells of the immune system217, 219-221,

while PLCβ4 expression is restricted to specific regions of the brain, e.g. cerebellum and retina222, 223. Of the PLCγ isozymes, PLCγ1 is ubiquitously expressed, particularly high in the

brain and lungs212, 224, while PLCγ2, although expressed quite widely throughout the body as

well, appears to be most abundant in cells of hematopoietic origin212, 225, 226. PLCε expression

is highest in the heart, with expression also high in both the lung and kidney227. Of the PLCδ

isozymes, δ1 is the most widely expressed, with highest expression in the heart, lung, skeletal muscle, brain, and testis212, 228; however, tissue levels of this isozyme appear to be relatively

low in comparison to PLCβ and PLCγ215. Surprisingly, relatively little is known with respect

to tissue distribution of PLCδ3 and PLCδ4 isozymes, although their expression has been reported in a number of rat tissues229. More recently identified PLCζ has been reported to be

sperm specific230, 231, while PLCη isozymes have thus far been exclusively found in

neurons195.

Differences in subcellular localization of PLC isozymes may also provide perspective with respect to diversity in individual functions of different isozyme families. Upon activation, every PLC isozyme generates two products, IP3 and DAG. Conventional dogma

suggests that cytosolic IP3 leads to increased intracellular calcium levels via receptor binding

at the endoplasmic reticulum, implying little possibility for downstream selectivity. More recently however, the identification of IP3 receptors at other cellular locations232, 233, as well

as the sequential formation of inositol polyphosphates within the cytosol 187-189 implies a

multitude of diversity for mediating numerous discrete downstream signaling events. DAG remains in the membrane and activates conventional and novel PKC isoforms. Activation of localized signaling complexes that includes a receptor, a PLC isozyme and an associated

PKC isozyme could also produce a mechanism for selective downstream signaling. Very recently, a signaling complex including EGF receptor, PLCγ1, and PKCε has been reported, providing direct evidence for such a hypothesis234. Therefore, differences in subcellular

localization are likely a key component for selectivity between isozyme-dependent downstream signaling pathways, a hypothesis supported by sequence variability in PLC PH domains, which are thought to mediate membrane binding as well as protein-protein interactions. The cellular localization of most PLCs has typically been reported to be primarily cytosolic in the resting state, and following receptor activation, they are then transiently recruited to the cell membrane via isozyme specific interactions of their PH domains with lipid membranes206, 235. For example, in wild-type Caco-2 cells PLCγ1 is

almost exclusively recovered in the cytosolic fraction; however, following EGF treatment and PLC activation, PLCγ1 is instead found entirely within the membrane fraction236. PLCγ also contains SH3 domains that bind proline-rich motifs on proteins; interestingly, ZO proteins contain proline-rich sequences that could bind and localize PLCγ to the tight junction. Although no reports exist that specifically colocalize PLCγ with junctional complexes, these isozymes have long been reported to colocalize with cortical actin filaments207, 237, which line the cell periphery and link the cytoskeleton directly to the tight

junction and neighboring cells. Recent studies further establish PLCγ interaction with cortical actin by demonstrating an SH2 mediated binding with the actin regulatory protein villin238,

239_{, a protein expressed exclusively in epithelial tissues, including the intestine.}

Evidence is emerging that PLCβ isozymes may be co-localized to the membrane with other signaling components. Most PLCβ isoforms are strongly associated with the membrane/particulate fraction, which is interesting since these enzymes bind phospholipids

weakly in vitro; therefore, other proteins204 and/or the cytoskeleton240 may organize these isozymes with other signaling proteins at the membrane. For example, recent reports suggest that scaffolding proteins containing PDZ domains play a significant role in the regulation of mammalian PLCβ isozymes. PDZ-containing proteins mediate the clustering of receptors and signaling molecules and thereby regulate agonist-induced signal transduction in polarized cells241. Interestingly, PDZ domains are found in ZO proteins of the tight junction, and a

recent study has reported binding of PLCβ3 to ZO-1 in rat fibroblasts209, providing direct

evidence that PLCβ isozymes may be localized to junctional complexes. Consistent with this recent report, another reported component of PLCβ signaling, Gα12, is co-localized with the

tight junction in Caco-2 and MDCK cells242, providing evidence for the likely presence of

PLCβ signaling complexes localized in the vicinity of tight junctions.

An abundance of evidence has also clearly demonstrated localization of PLCβ isozymes to the nucleus, likely mediated via a nuclear localization signal sequence in their unique carboxy terminal extension208_{, where they play a key role in cell proliferation and}

differentiation243-246; their presence at the nuclear membrane indicates diverse roles for these

isozymes that may be mediated by differential regulation, as there is no evidence that G- protein subunits that regulate these isozymes at the cell membrane are expressed in the nuclear compartment. The role of PLCβ isozymes in nuclear signaling has been recently reviewed247, 248. While all four members of the PLCβ family have been localized both inside

and outside the nucleus, the subcellular localization of PLCδ isozymes appears to be more specific. PLCδ4 is thought to be primarily nuclear249, while PLCδ1 and PLCδ3 possess and

nuclear export sequence within the EF-hand domain, implying they may shuttle between the nucleus and cytoplasm. Further, PLCδ1 has been reported to be mostly cytoplasmic, while

PLCδ3 is found in membrane fractions250-253. Although neither PLCγ isozyme contain a

known nuclear signal sequence, they too have been reported to be localized to nuclei of certain cell types243.