Often, PDZ domains are mentioned to repeatedly occur within one protein chain.
However, it seems that this property is less general than thought as about 75 % of all human PDZ domain-containing proteins (short ”PDZ proteins“) contain only one PDZ domain (see Figure 3.5). This prevailing view may originate from a few intensely studied multiple PDZ proteins that are recurrently mentioned in the next paragraphs.
PDZ domains co-occur with several other types of globular domains in proteins, e.g.
the membrane-associated guanylate kinase (MAGUK) family of proteins contains a conserved triplet of domains consisting of a PDZ, an SH3 and a guanylate kinase (GK) domain [107]. This family of proteins has currently 16 members including the DLG proteins and the ZO proteins. The MAGI proteins lack the SH3 domain and present the PDZ and GK domain in inverted order. Thus, they should not be consid-ered as MAGUKs contrary to assertions in the published literature [108]. Other types of domains that frequently co-occur with PDZ domains in proteins are ankyrin, LIM, L27, C2, PH, WW, DEP, and LRR domains [80]. Some enzymatic activities have been observed in PDZ proteins, e.g. serine-threonine kinase, phosphatase, protease, guanine nucleotide exchange factors (GEFs) and GTPase activities [80].
0 5 10 15
1 2 3 4 5 6 7 8 9 10 11 12 13
Frequency of occurrence
Number of PDZs per protein 100
105
Figure 3.5. Distribution of numbers of PDZ domains per protein in the human proteome.
The data used to create the plot has been derived from [56].
Clearly, the multidomain character of PDZ proteins largely determines the functions of these proteins. They have been found to be mainly cytosolic proteins that assist in the assembly and localisation of protein complexes that participate in intracellular signalling pathways [6]. As it seems that these proteins serve as an initial platform for protein complex assembly, the term scaffold has been recurrently used to describe this important functionality of PDZ proteins. In the following, I summarise important biological processes that are regulated by PDZ proteins with a strong focus on cell polarity establishment and maintenance. Names of PDZ proteins are highlighted with bold letters.
One of the most important insights that I obtained during my PhD consisted of the fundamental and diverse roles that cell polarity plays in multicellular organisms.
Every cell that is not a completely undifferentiated stem cell is very likely to be polar, e.g. will exhibit an asymmetric distribution of molecules and a shape different from a sphere. Cell polarity in its various forms is important for asymmetric cell division, neuronal transmission, cell migration, immunological responses, and establishment of tissue layers, such as epithelia and endothelia. This non-exhaustive list of functions of cell polarity illustrates its importance and remarkably, in all these processes, PDZ proteins play essential roles.
Establishment and maintenance of cell polarity is highly dependent on the dynamic remodelling of the actin cytoskeleton that is regulated by small Rho GTPases [109].
PDZ proteins have been shown to interact with various members of the G protein cycle (see Figure 3.6), including G-protein coupled receptors (GPCRs), GTPases, and GEFs (see section 3.2.5 and own findings presented in chapter 9). PDZ proteins seem to bring components of the G protein cycle together at precise locations beneath plasma
membranes where they allow for the activation of intracellular signalling pathways in response to incoming stimuli at receptors.
GEF GEF
GEF
Effector RhoGTPase GTP RhoGTPase
GTP
GAP PO43–
GTPhydrolysis GDP-GTP
Exchange GTP
GDP
Sequestration
in cytosol GDI
GDI
GDI
Downstream signalling Phospholipids
Plasma membrane
Cytosol
RhoGTPase GDP RhoGTPase
GDP
RhoGTPase GDP
Figure 3.6. The G protein cycle of small Rho GTPases. G proteins are important signalling molecules. They are GTPases that are inactive when bound to GDP (guanosine diphosphate).
GEFs (guanine nucleotide exchange factors) activate GTPases by exchanging GDP with GTP (guanine triphosphate). Depending on associated effector proteins, GTPases can function in specific downstream signalling pathways. GTP-activating proteins (GAPs) can accelerate hydrolysation of GTP to GDP by GTPases, which leads to their inactivation. Guanine nucleotide dissociation inhibitors (GDIs) can retain GTPases in their inactive state. The figure has been extracted from [109].
3.3.1. Epithelial apical-basal cell polarity
Epithelial cell layers function as barriers between compartments, e.g. the inside and outside of an organism, and allow for selective transport of molecules from one side to the other [110]. The apical side is oriented towards the outside whereas the basal side is oriented towards the inner side. The basal side of the cell is attached to the
extracellular matrix. The cellular space between the apical and basal part is called the lateral side and is the area of contact with neighbouring cells of the same cell layer (see Figure 3.7). Thus, orientation of the apical–basal axis in an epithelial cell is defined by its environment. Several sites of cell–cell contact ensure the proper anchorage of an epithelial cell within the cell layer. Adherens junctions regulate cell–cell adhesion by providing the mechanical link between cells. They contain cadherins and catenins and are linked to the cytoskeleton via the proteinAfadin (AF6)[110]. Tight junctions are located above adherens junctions and mark the border between the apical and lateral domain of a cell (see Figure 3.7). Tight junctions create a diffusion barrier for soluble molecules between cells and preclude an intermixture of components of the apical and lateral membrane. They contain occludins, junction adhesion molecules (JAMs) and claudins, and are mainly organised by theZOfamily of proteins (see Figure 3.8) [111].
MAGI proteins have been shown to be abundant at tight junctions where they link the JAMs to the atypical protein kinase C (aPKC) signalling pathway [112].
Crumbs-3
PAR6 aPKC PALS1
TJ PATJ
AJ
PAR3
Phosphorylation
LGL1/2
DLG1 Scribble Mutual
exclusion
ApicalLateralBasal
Figure 3.7. Epithelial cell polarity and organisation of polarity complexes. The apical, lateral and basal part of a polar epithelial cell are indicated to the right. TJ = tight junction, AJ = adherens junction. Three polarity complexes are the main regulators of epithelial cell polarity: the Crumbs, Par3, and SCRIB complex. They influence each others cellular localisation. The figure has been adapted from [109].
Establishment of apical–basal cell polarity requires both cadherin-dependent cell-cell adhesion and adhesion to the extracell-cellular matrix [110]. The protein complexes that organise epithelial apical–basal cell polarity are conserved from the fly (where they have been discovered) to worm and human [111]. Asymmetric concentration of
Figure 3.8. Organisation of tight junctions. PDZ proteins like the ZO-family of proteins and AF-6 are mainly responsible for protein complex organisation beneath tight junctions and their connection to the cytoskeleton. The figure has been taken from [113].
phosphatidylinositides may be the initiator of a first localisation of the polarity com-plexes [110]. The Par3 complex (composed of Par3, Par6, and aPKC) as well as the Crumbs complex (composed of MAGUK p55 subfamily member 5 (MPP5), pals1-associated tight junction protein (PATJ), and LIN7) are localised api-cally to places where tight junctions will be formed [110]. The SCRIB complex (com-posed of SCRIB, Lgl1/2, and DLG1) localises to the lateral membrane (see Figure 3.7). The PDZ protein members of these complexes engage in numerous PDZ domain-mediated protein interactions that organise their localisation, the assembly of the tight and adherens junctions and their link to the cytoskeleton [110, 111]. Knockout experiments of members of the polarity complexes usually revealed only very mild phenotypes making it difficult to study their precise functions. Probably, the high functional redundancy within the polarity complexes confers robustness to the system that regulates cell polarity.
3.3.2. Polarisation in neurons
Neurons are highly polarised cells although it is a very different polarisation from the apical-basal polarity observed in epithelial cells. The asymmetry of neurons is dis-played by presynaptic and postsynaptic sites that are formed at the axon terminal and dendrites, respectively [109]. These sites contain distinct ensembles of proteins that ensure the directional transmission of action potentials [111]. The spatially restricted activation of the Par3 complex together with the protein T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) has been shown to be important for the process of axon specification [109].
PDZ domains have been first discovered in the protein PSD-95 (see section 3), a main actor in the postsynaptic density. PSD-95 together with other PDZ pro-teins such as GRIP1, PICK1, and DLG1 regulate the clustering and localisation of receptor channels (e.g. AMPA and N-methyl-D-aspartate (NMDA) receptors) at postsynaptic sites and organise a dense network of protein interactions that link the membrane-anchored protein complexes to downstream signalling pathways [6, 80].
3.3.3. T-cell polarity
T-cells that circulate in blood vessels and lymphatic vessels have a round morphology.
Following stimulation by external chemokines, they polarise to be able to migrate to-wards inflamed tissue or to mediate cell–cell interactions (e.g. with antigen-presenting cells). T-cells polarise along the anterior-posterior axis. SCRIBand DLGproteins are localised to the rear of the polarising cell to initiate uropod formation (membrane protrusion at rear of T-cells that is involved in their activation and migration) [109].
The Par3 protein complex is localised to the front of the T-cell where it initiates lamellipodium formation (sheet-like cellular protrusion that is enriched in actin) [109].
An immunological synapse that is formed between a polarised T-cell and an antigen-presenting cell, constitutes a transient and adhesive contact. During immunological synapse formation, polarity proteins such asDLGandSCRIBhave been observed to redistribute from the uropod to the immunological synapse. Knockdown of SCRIB led to defects in polarisation indicating the importance of PDZ proteins in immuno-logical synapse formation [109].
3.3.4. Cell migration
Cell migration can be observed in embyonic and adult organisms as well as in patho-logical situations such as inflammation and cancer. Neurons migrate along glial cells during brain development, epithelial cells migrate during tissue morphogenesis and for maintaining the skin, and tumour cells migrate during metastasis. In order to migrate, cells have to be polarised along a front-rear axis. Rho GTPases and components of the Par3, SCRIB and Crumbs complexes have been demonstrated to jointly reg-ulate front-rear polarisation, chemotactic migration and wound-healing in epithelial cells [109].
3.3.5. Asymmetric cell division
Asymmetric cell division occurs during embryonic development and in adult organ-isms (e.g. maintenance of stem-cell populations) and requires asymmetric distribution of polarity proteins prior to cell division [109]. In neuroblasts, the apical cortex is enriched for the Par3complex whereas theSCRIBcomplex regulates the alignment of the mitotic spindle along the apical-basal axis. Cell-fate determinants accumulate according to the distribution of the polarity complexes and allow for the creation of two different daughter cells [109].
3.3.6. Cell polarity and tumourigenesis
Growth and proliferation of a cell is tightly controlled by its anchorage within a tissue.
If a cell looses its cell polarity regulators, it will loose its tight connections to neigh-bouring cells. Most malignant tumor cells have lost some stages of polarity. Thus, they can escape from normal proliferation control and display increased migratory capacity [109, 114]. Consistently, SCRIB has been termed a tumour suppressor for its function in apical-basal cell polarity maintenance [115]. Nevertheless, given the intertwined relationship of the polarity complexes and their implications in a very diverse range of biological processes, their role in tumourigenesis can be oncogenic or suppressive depending on the context [114, 116].
3.3.7. Apart from cell polarity
Of course, PDZ proteins also perform functions that are (at least not directly) linked to cell polarity. NHERF1 has been in the focus of many studies for its implication in membrane protein activity and trafficking [6]. No SH2 domains nor tyrosine kinase activities have been found in proteins together with PDZ domains [80]. However, PDZ proteins are frequently observed as adaptors for tyrosine kinase receptors, such as the PDZ protein Erbin that recognizes a PBM in the receptor tyrosine-protein kinase ERBB2 [80]. The PDZ protein PATJhas been highly investigated for its scaffolding function in the phototransduction pathway in the eye ofD. melanogaster [69,117]. The PDZ protein Harmonin is essential for proper mechano-transduction in the inner ear sensory hair cells. Defects in Harmonin cause the Usher syndrome, a disease characterised by deafness and blindness [118].