Phosphatidylinositol (PI) is the precursor lipid for the synthesis of PI 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane (PM) and is sequentially phosphorylated by the lipid kinases, PI 4-kinase and phosphatidylinositol 4-phosphate (PI4P)-5-kinase. Receptor-mediated hydrolysis of PI(4,5)P2 takes place at the PM but PI resynthesis occurs at the endoplasmic reticulum (ER). Thus PI(4,5)P2 resynthesis requires the reciprocal transport of two key intermediates, phosphatidic acid (PA) and PI between the ER and the PM. PI transfer proteins (PITPs), defined by the presence of the PITP domain, can facilitate lipid transfer between membranes; the PITP domain comprises a hydrophobic cavity with dual specificity but accommodates a single phospholipid molecule. The class II PITP, retinal degeneration type B (RdgB)α is a multi-domain protein and its PITP domain can bind and transfer PI and PA. In Drosophila photoreceptors, a well-defined G-protein-coupled phospholipase Cβ (PLCβ) signalling pathway, phototransduction defects resulting from loss of RdgBα can be rescued by expression of the PITP domain provided it is competent for both PI and PA transfer. We propose that RdgBα proteins maintain PI(4,5)P2 homoeostasis after PLC activation by facilitating the reciprocal transport of PA and PI at ER–PM membrane contact sites.

Introduction

Phospholipase C (PLC) activation by cell surface receptors mediates the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate [PI(4,5)P2] to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG). Depletion of PI(4,5)P2 at the plasma membrane (PM) affects several processes including cytoskeletal dynamics, ion channel regulation and endocytosis and therefore rapid replenishment of PI(4,5)P2 is essential to maintain cellular function. To resynthesize PI(4,5)P2, DAG is first phosphorylated to phosphatidic acid (PA) at the PM. PA is the substrate for CDP–DAG synthase (CDS) to produce CDP–DAG which is converted into PI by PI synthase (PIS) at the endoplasmic reticulum (ER; Figure 1) [1]. At the PM, PI can be sequentially phosphorylated by the resident PI 4-kinaseα and  phosphatidylinositol 4-phosphate (PI4P)-5-kinase to PI(4,5)P2 [2]. The topological arrangement of these enzymes requires the transfer of PI and PA in opposite directions between two membrane compartments, the PM and the ER (Figure 1). Although the enzymes involved in these steps of lipid metabolism are well characterized, the major question yet to be resolved is the mechanism of PI and PA transfer between the two membrane compartments. This question was raised in 1975 by Michell [3] when the ‘PI cycle’ was suggested to be important in cellular function and predicted that lipid transporters would move the lipids between the membranes. A lipid transport protein capable of transporting PI had just been identified in 1974 supporting this concept [4].

Topological organization of the ‘PI cycle’

Figure 1
Topological organization of the ‘PI cycle’

Agonist-dependent activation of PLC at the plasma membrane hydrolyses PI(4,5)P2 to DAG which is rapidly converted into PA. PA can also be generated by PLD upon receptor activation. To resynthesize PI, PA has to be transported to the ER and after its synthesis, PI has to be transported to the PM where the two lipid kinases work in sequence to replenish the PI(4,5)P2 levels. The molecular machinery that moves the PA and PI between the ER and the plasma membrane is suggested to be a member of the PITP family of proteins. Abbreviations: DGK, DAG kinase; GPAT, glycero-phosphate acyl transferase; LPA, lyso-PA; LPAAT, lyso-PA acyl transferase; PI4Kα; PI 4-kinase; PI4P5K, PI4P 5-kinase.

Figure 1
Topological organization of the ‘PI cycle’

Agonist-dependent activation of PLC at the plasma membrane hydrolyses PI(4,5)P2 to DAG which is rapidly converted into PA. PA can also be generated by PLD upon receptor activation. To resynthesize PI, PA has to be transported to the ER and after its synthesis, PI has to be transported to the PM where the two lipid kinases work in sequence to replenish the PI(4,5)P2 levels. The molecular machinery that moves the PA and PI between the ER and the plasma membrane is suggested to be a member of the PITP family of proteins. Abbreviations: DGK, DAG kinase; GPAT, glycero-phosphate acyl transferase; LPA, lyso-PA; LPAAT, lyso-PA acyl transferase; PI4Kα; PI 4-kinase; PI4P5K, PI4P 5-kinase.

In 1993, we identified a soluble 35-kDa protein, PI transfer protein (PITPα) as capable of supporting Ins(1,4,5)P3 production in permeabilized cell preparations [57]. PITPα has dual specificity; it can accommodate either a PI or a phosphatidylcholine (PC) molecule within its hydrophobic cavity [8,9]. The issue of PA transport has remained unresolved until recently. A previous study, again in permeabilized cells, indicated that PA transport from the ER was not mediated by soluble proteins or by vesicular transport and we suggested that it could be protein-mediated at sites of close membrane contact by membrane proteins [10]. In 2012, we identified class II PITPs as PA and PI transport proteins [11], unlike PITPα and PITPβ which transport PI and PC. In yeast, a PA transporter that functions in mitochondria, Ups1 in complex with Mdm35, was also identified in 2012; Ups1 and PITPα share structural homology but not sequence homology [1214]. In the present review, we focus on members of the class II PITP family that can reciprocally transfer PI in exchange for PA at membrane contact sites during PLC signalling (Figure 2).

Proteins with a PITP domain in humans, Drosophila and C. elegans

Figure 2
Proteins with a PITP domain in humans, Drosophila and C. elegans

The PITP domain is ∼260 amino acids; class I PITPs comprise the PITP domain only whereas RdgBα proteins are multi-domain proteins. The ‘FFAT’ motif (EFFDAxE) binds to ER-localized VAP proteins [50]. Mammals including humans contain five proteins with a PITP domain whereas Drosophila and C. elegans contain three proteins. The proteins are grouped into class I and II based on sequence; class I PITPs bind and transfer PI and PC whereas class II PITPs bind and transfer PI and PA.

Figure 2
Proteins with a PITP domain in humans, Drosophila and C. elegans

The PITP domain is ∼260 amino acids; class I PITPs comprise the PITP domain only whereas RdgBα proteins are multi-domain proteins. The ‘FFAT’ motif (EFFDAxE) binds to ER-localized VAP proteins [50]. Mammals including humans contain five proteins with a PITP domain whereas Drosophila and C. elegans contain three proteins. The proteins are grouped into class I and II based on sequence; class I PITPs bind and transfer PI and PC whereas class II PITPs bind and transfer PI and PA.

Introduction to the PITP family

The PITP family has five members in the mammalian genome subdivided into two classes based on sequence analysis (Figure 2). Analysis of the lipid transfer and binding activities of the various PITP domains establish that class I PITP are PI and PC transfer proteins whereas class II PITPs are PI and PA transfer proteins (Figure 3). Class I comprises the single-domain PITPs (α and β) and class II comprises the retinal degeneration type B (RdgB) proteins. The RdgB family is so named as the founding member of this class was first identified as the retinal degeneration B mutant in Drosophila [15]. The PITPs are defined by the presence of the PITP domain [Pfam: IPtrans (PF02121)]; this domain can bind and facilitate the exchange of PI between membrane compartments without a requirement for ATP. The hallmark of this domain is four amino acid residues present on two β-strands. These residues (Thr59, Lys61, Glu86 and Asn90 using mouse numbering) are conserved in the majority of the PITP sequences found in the sequence database across different species. Single point mutations in any of these residues renders them inactive for PI binding or transfer [9]. There are three RdgB proteins in the mammalian genome, two of which are large multi-domain proteins containing the DDHD and LNS2 domain. The DDHD domain is 195 amino acids and is also present in other proteins including DDHD1 and DDHD2 (KIAA0725p) that possess PA-PLA1 activity and p125 (Sec 23-interacting protein). This domain has been shown to bind PI4P in vitro [16,17]. The LNS2 domain is 130 amino acids and is also found in lipins (PA phosphatase; although the motif required for phosphatase activity is not conserved) and has been shown to bind PA [18]. These proteins possess six hydrophobic regions and thus endogenous proteins, both in mammals and in flies, are membrane-associated [1921]. The proteins can be solubilized at pH 10 or high salt but are not solubilized with Triton X100 suggesting that the proteins are peripheral and are potentially associated with the cytoskeleton. The third RdgB protein, RdgBβ, consists of the PITP domain followed by an unstructured eighty amino acid N-terminal extension which is phosphorylated at two sites to form a 14-3-3 binding site [22]. In addition to binding 14-3-3, the PITP domain binds an integral adaptor protein, ATRAP (angiotensin II receptor-associated protein) [23].

Lipid transfer activities of the PITP domains of class I and class II RdgB proteins

Figure 3
Lipid transfer activities of the PITP domains of class I and class II RdgB proteins

Human PITPα (black circles), human RdgBβ (green triangles) and the PITP domain of human RdgBαI (yellow triangles) and Drosophila RdgBa (red circles) were examined for PI, PC and PA transfers activity. PITPα transfers PI and PC whereas RdgB proteins transfer PI and PA. The y-axis represents the amount of labelled lipid transferred to an acceptor compartment as a percentage of the total input present in the donor compartment.

Figure 3
Lipid transfer activities of the PITP domains of class I and class II RdgB proteins

Human PITPα (black circles), human RdgBβ (green triangles) and the PITP domain of human RdgBαI (yellow triangles) and Drosophila RdgBa (red circles) were examined for PI, PC and PA transfers activity. PITPα transfers PI and PC whereas RdgB proteins transfer PI and PA. The y-axis represents the amount of labelled lipid transferred to an acceptor compartment as a percentage of the total input present in the donor compartment.

Expression pattern of PITPs

In order to understand the function of the PITPs, establishing their cellular expression pattern and localization is vital. In mammals, PITPα is highly enriched in the brain but is found in virtually all tissues [24]. Likewise, PITPβ is enriched in liver, lung and muscle but is present at the protein level in most tissues examined. When cytosols from different tissues have been fractionated by size exclusion chromatography, a single peak of PI transfer activity is found which is due to the combined presence of PITPα and PITPβ [25]. RdgBβ, on the other hand, is enriched in the heart and in the brain but when cytosols prepared from these tissues are analysed for PI transfer activity, a peak of activity associated with RdgBβ is not observed. This suggests that RdgBβ functions locally at membrane contact sites and is probably recruited to membranes by ATRAP [22]. The endogenous localization of the two multi-domain proteins, RdgBαI and RdgBαII, has been analysed in only a few cases. RdgBαI (also known as PITPNM1) is found in inner hair cells of the cochlea, the retina and in specific areas of the brain [26,27]. RdgBαII expression is highly restricted; it is mainly found in the retina and the dentate granular cells in the hippocampus [27,28]. In the retina, it is found discretely in the GABAergic amacrine cells and the ganglion cell layer [29].

The Drosophila and Caenorhabditis elegans genomes only contain a single RdgBα protein. In Drosophila, the protein is enriched in photoreceptors but also other specific regions of the brain [30]. C. elegans RdgBα also shows specific expression in the nervous tissue [31]. It is present in a subset of neurons including ASER and AWC. (C. elegans possess 302 neurons and all the neurons have been named and their positions mapped [32]).

Analysis of Drosophila RdgBα in phototransduction

Sensory transduction in Drosophila photoreceptors relies on G-protein coupled PLC activation. Rhodopsin, together with Gq and PLCβ (NorpA) are concentrated in the microvillar membranes whereas Drosophila RdgBα (Dm-RdgBα) localizes to the subrhabdomeric cisternae (SRC) [30,33]. Light induces robust activation of PLCβ resulting in the consumption of PI(4,5)P2 [34]. In the absence of Dm-RdgBα, photoreceptors show a defective electrical response to light along with retinal degeneration. The retinal degeneration phenotype of the rdgB mutant requires ongoing light-activated PLC-mediated hydrolysis of PI(4,5)P2. Thus rearing flies in the dark or norpA mutants that lack a functional PLCβ is protective [35]. In contrast, retinal degeneration is accelerated in flies that express constitutively-active dGq. Retinas of rdgB;dGq double mutants degenerate even in the dark and is PLC-dependent [36]. PIS is required for a key step during PI(4,5)P2 regeneration and overexpression of PIS is reported to suppress the retinal degeneration of rdgB and cds mutants [37]. The rdgB mutants show reduced amounts of PI(4,5)P2 in the rhabdomeres even before they are exposed to any light. Additionally, the time-course of PI(4,5)P2 resynthesis after light exposure is delayed and is accompanied by an increase in PA levels [21].

Expression of a PITP domain competent to transfer both PI and PA is sufficient to rescue both retinal degeneration and the electrophysiological light response [21]. Class I PITPs, either mammalian or Drosophila, which transfer PI and PC but not PA, are unable to facilitate rescue. Mutation of the residues required for binding and transferring PI (Thr59, Lys61, Asn90) in either the PITP domain or the full-length protein are also unable to rescue function. Docking of the class I PITPs to membranes requires two tryptophan residues and mutation of these residues disrupts membrane docking although their lipid-binding capacity is unimpaired [9,25]. In class II PITPs, this motif is YW and mutation of this motif in the full length RdgBα protein is unable to rescue retinal degeneration and the electrical response. Together these data provide compelling genetic evidence to support the concept that the rdgB mutant phenotype is due to the inability to replenish PI(4,5)P2 levels following stimulation by light and the mechanism of RdgBα function in the restoration of PI(4,5)P2 levels depends on PI and PA transfer. Thus reciprocal transfer of PA and PI from the PM to SRC and vice versa by the PITP domain of RdgBα provides an elegant solution to the maintenance of PI(4,5)P2 homoeostasis during PLC signalling (Figure 4).

Reciprocal PI and PA transfer by RdgBα

Figure 4
Reciprocal PI and PA transfer by RdgBα

RdgBα is localized at the ER membrane and the PITP domain of RdgBα can transfer PI and PA between the ER and PM at sites of close membrane contact. In Drosophila photoreceptors, the gap between the rhabdomere (PM) and the SRC is estimated to be 10 nm. The FFAT motif in RdgBα can bind to the integral VAP protein present at the SRC.

Figure 4
Reciprocal PI and PA transfer by RdgBα

RdgBα is localized at the ER membrane and the PITP domain of RdgBα can transfer PI and PA between the ER and PM at sites of close membrane contact. In Drosophila photoreceptors, the gap between the rhabdomere (PM) and the SRC is estimated to be 10 nm. The FFAT motif in RdgBα can bind to the integral VAP protein present at the SRC.

Analysis of RdgBα function in C. elegans

In the worm, the orthologue of RdgBα, Ce-RdgBα (also referred to as PITP-1) is required for sensory transduction in specific neurons including ASER, AWC and ASH that are involved in the gustation, olfaction and osmo-sensation respectively. This recapitulates the enrichment of Dm-RdgBα in sensory organs of the adult head. Individual phenotypes resulting from loss of Ce-RdgBα can be restored when Ce-RdgBα is re-expressed in specific neurons [31]; as in Drosophila, reconstitution of the Ce-RdgBα mutant with the PITP domain alone is sufficient to rescue mutant phenotypes supporting the notion that the PITP domain contains an important functional activity of this protein.

Ce-RdgBα is localized in the axons of the sensory neuron, ASER, involved in salt attraction. The reduced salt attraction seen in Ce-RdgBα mutants could be suppressed by mutations in DAG kinase, which causes accumulation of DAG indicating that DAG elevation is required for synaptic transmission. These results suggest that Ce-RdgBα maintains PI(4,5)P2 levels required for the production of DAG via PLC. The PLC in question has been identified as PLC-epsilon, as a mutation in this enzyme also alters salt preference [38]. Collectively these findings imply that the function of Ce-RdgBα occurs in the context of PLC signalling and supports lipid turnover during the PI(4,5)P2 cycle.

Although Ce-RdgBα is essential for a number of sensory behaviours, locomotion and egg-laying are unaffected indicating a highly specific requirement. Both locomotion and egg laying are defective in egl-8 PLCβ mutants and egl−30 Gqα mutants indicating that these processes are dependent on PLC signalling [39] but not Ce-RdgBα. There are two other PITPs in C. elegans and could potentially participate in these PLC-dependent processes. Their lipid transfer activities have not been examined.

RdgBα is present at membrane contact sites

In Drosophila photoreceptors, Dm-RdgBα is found at the membranes of the SRC which lies underneath the rhabdomeric PM at the bases of the photoreceptive microvilli [30,33]; the SRC is equivalent to the ER and lies 10 nm from the microvillar PM. The N-terminal PITP domain is followed by a disordered region of 120 amino acids before the ‘FFAT’ (two phenylalanines in an acidic tract) motif (that would anchor the protein to the ER by binding to Dm-VAP; Figure 4). A fully extended amino acid is 3.3 Å (1 Å=0.1 nm) and therefore a disordered region of 120 amino acid could in principle span 40 nm, more than sufficient for the PITP domain to make contact with the PM. Thus the PITP domain of RdgBα would be able to reach out to the PM (Figure 4). The orientation of the LNS2 and DDHD domains is not known however.

Recent studies in mammalian cultured cell lines also suggest that mammalian RdgBαI (also known as PITPNM1/Nir2) and mRdgBαII (also known as PITPNM2/Nir3) are present at ER–PM contact sites [4043]. These studies suggest that overexpressed mRdgBα is recruited from the cytosol to the PM by the LNS2 domain  binding to PA generated by agonist-activated PLC. To replenish the PI(4,5)P2 pool, the PITP domain was also required. Although the conclusions from the studies in Drosophila, C. elegans and the cultured cell lines are broadly in agreement, there are differences.

Firstly, there is conflicting data concerning RdgBαI localization in cultured cell lines. Since some of these studies relied on overexpression, this may affect the localization. In one study, RdgBαI was localized to the Golgi whereas two other studies found it to be cytosolic [18,41,42]. In the study by Kim et al. [18], both endogenous and myc-tagged RdgBα was Golgi-localized and upon stimulation with EGF, RdgBα translocated to the PM [18]. The PITP domain determined Golgi-localization, whereas translocation to the PM required the LNS2 domain. The LNS2 domain was found to bind PA in vitro and it was suggested that the translocation was due to a rise in PM PA. In contrast, Liou and colleagues [41] have reported that elevation of cytosol Ca2+ triggers the translocation of E-Syt to ER–PM junctions and this subsequently recruits cytosolic RdgBαI to the junctions. (E-Syt1 is a transmembrane protein localized to the ER which contains an SMP domain and five C2 domains. One of the C2 domains can bind to PM PI(4,5)P2 in a Ca2+-dependent manner [44]). Recruitment to ER–PM junctions was independent of PI binding to the PITP domain but PI binding was required for PI(4,5)P2 replenishment.

The study by Kim et al. [42] has also examined the role of RdgBαI in maintaining PI(4,5)P2 levels after PLC activation. Their study was conducted in human embryonic kidney (HEK)293 cells stably expressing the angiotensin II receptor. They also report that RdgBαI is mainly cytosolic with some localization at the ER. No Golgi localization was observed. In RdgBαI knockdown cells, they report less agonist-stimulated DAG, but observe a larger increase in PA at the PM. A decrease in the synthesis of the intermediate CDP–DAG was also seen. They also observe that RdgBαI localizes to ER–PM contact sites upon stimulation and is dependent on the FFAT motif binding to VAP proteins. Using an experimental protocol that monitored the removal of PA from the PM, they found that upon overexpression of RdgBαI, PA removal was faster compared with the controls. Removal of PA was dependent on the PITP domain. Most interestingly, mutation of Thr59 to either E or A prevented the stimulatory effect on PA clearance. Although not shown for human RdgBαI, it has been previously shown that mutation of Thr59 to A or E in either class I or class II inhibits PI transfer but not PA transfer activity of Dm-RdgBα PITP domain [9,21]. It is therefore puzzling why mutation of Thr59 disrupts PA clearance. It would be interesting to examine PA transfer activity of these mutants. For RdgBαI to associate with membranes, they suggest that the region that connects the LNS2 domain with the DDHD domain contains a short sequence that bears resemblance to the DAG-binding part of the C1 domain. This domain together with the LNS2 was required for PM association to DAG.

Concluding remarks

Studies from both model organisms, Drosophila and C. elegans, clearly indicate that RdgBα is part of the PLC signal transduction cascade where the PITP domain is central to its function and is required for the replenishment of PI(4,5)P2 after its degradation by PLC. However, in these model organisms, RdgBα is required in highly-specific situations. In the case of Drosophila, it is required for phototransduction whereas in C. elegans it is obligatory for sensory transduction in a set of defined neurons. In mice, the requirement for RdgBαII also appears to be highly-specific. RdgBαII mutants display defects in circadian photoentrainment and the pupillary light response but only in dim light and not in bright light [29]. In bright light, the intrinsically photosensitive retinal ganglion cells (ipRGC) use melanopsin to activate PLCβ4 but RdgBαII is not required. (It is not clear if RdgBαI is expressed in these cells). PLC signalling in mammals is ubiquitous and there are 13 PLCs in the human/mouse genome. Gene knockouts of individual PLCs provide a variety of strong phenotypes including epilepsy, embryonic lethality, etc. [45]. In contrast, mice null for RdgBαI and RdgBαII show only very subtle phenotypes; mice are grossly normal and fertile suggesting that either RdgBα functions in specific neuronal circuits such as sensory transduction, learning and memory [26,29] or other PITPs can compensate. (It has been previously reported that RdgBαI-null mice could not be generated [28] but a more recent study report otherwise [26]). There are five PITP proteins in the mammalian genome and whereas both class I and II PITPs can transfer PI, only class II PITPs can transfer PA. Class I PITPs are ubiquitously expressed but this leaves open the question of PA transfer from the PM to ER. Is it possible that not all PLC signalling cascades need a PITP or alternatively class I PITP can also supply PI for PI(4,5)P2 homoeostasis? Maybe, replenishment of PI at the PM from the much larger pool of the ER is more critical than PA removal. This question can be addressed by deletion of all the PITP proteins.

PLC activation by cell surface receptors mediates the hydrolysis of PI(4,5)P2 to Ins(1,4,5)P3 and DAG. Although the initial focus of the PLC pathway was on the generation of the second messengers, Ins(1,4,5)P3 and DAG, PLC activation can also acutely regulate the levels of PI(4,5)P2. In some cases, PI(4,5)P2 depletion is more relevant than the second messengers.

For example, both phototransduction in Drosophila and inhibition of KCNQ2/3 potassium channels by activation of muscarinic cholinergic receptors requires depletion of PI(4,5)P2. In the case of phototransduction, depletion of PI(4,5)P2 regulates Ca2+ entry through TRP (transient receptor potential) channels and in the case of KCNQ2/3, depletion of PI(4,5)P2 is required for closure of the potassium channels. This demands intense hydrolysis but also requires rapid replenishment to avoid any impact on endocytosis or on the actin cytoskeleton which are sensitive to PI(4,5)P2 levels.

The level of PI(4,5)P2 depletion that occurs during GPCR signalling is dependent on the number of receptors activated [4648]. If Ins(1,4,5)P3/DAG production is the required response, the number of receptors that need to be activated can be small; and PI(4,5)P2 depletion will be minimal. The amount of Ins(1,4,5)P3 required to maximally release cytosol Ca2+ can be saturated with minimal depletion of PI(4,5)P2. On the other hand, if PI(4,5)P2 depletion is required for regulation of ion channels as discussed above, then a higher density of receptor activation is required [47,48]. Thus full amplitude calcium responses can be elicited with minimal PI(4,5)P2 depletion. Alternatively, if inhibition of potassium channels is required, then substantial PI(4,5)P2 hydrolysis is required.

In addition to GPCRs, receptor tyrosine kinases also activate PLC, in this case, the PLCγ family. Again, EGF receptors can also induce significant PI(4,5)P2 hydrolysis, in this case, to release the actin-binding protein, cofilin. Cofilin is restrained at the PM by being bound to PI(4,5)P2. Upon PI(4,5)P2 hydrolysis, cofilin is released and can now bind and sever F-actin for restructuring the actin cytoskeletal network [49]. These examples demonstrate the versatility of PI(4,5)P2, not only as a provider of second messengers, but PI(4,5)P2 functioning as a regulator as an intact lipid.

The difference in PI(4,5)P2 hydrolysis depending on the required output raises the important question: are there special mechanisms in place when PI(4,5)P2 depletion is the required response {as opposed to the production of Ins(1,4,5)P3/DAG} for replenishing the PI(4,5)P2 levels? Do PITP proteins become essential only when the amount of PI(4,5)P2 hydrolysed is high as this places a burden on the cell and rapid replenishment is required? When PLC stimulation which occurs at the physiological level for increasing Ca2+, PI(4,5)P2 hydrolysis will be limited and therefore replenishment of PI(4,5)P2 may not require specialized mechanisms. Thus we would propose that PITP proteins become essential mainly during intense PLC signalling.

Funding

This work was funded by the Biotechnology and Biological Sciences Research Council and the British Heart foundation [grant number 044/25411].

Abbreviations

     
  • ATRAP

    angiotensin II receptor-associated protein

  •  
  • CDS

    CDP–DAG synthase

  •  
  • DAG

    diacylglycerol

  •  
  • EGF

    epidermal growth factor

  •  
  • ER

    endoplasmic reticulum

  •  
  • FFAT

    two phenylalanines in an acidic tract

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • Ins(1,4,5)P3

    inositol 1,4,5-trisphosphate

  •  
  • PA

    phosphatidic acid

  •  
  • PC

    phosphatidylcholine

  •  
  • PI(4,5)P2

    phosphatidylinositol 4,5-bisphosphate

  •  
  • PI

    phosphatidylinositol

  •  
  • PI4P

    phosphatidylinositol 4-phosphate

  •  
  • PI4P5K

    PI4P 5-kinase

  •  
  • PIS

    phosphatidylinositol synthase

  •  
  • PITP

    phosphatidylinositol transfer protein

  •  
  • PL

    phospholipase

  •  
  • PLC

    phospholipase C

  •  
  • PM

    plasma membrane

  •  
  • RdgB

    retinal degeneration type B

  •  
  • SRC

    subrhabdomeric cisternae

Signalling 2015: Cellular Functions of Phosphoinositides and Inositol Phosphates: Held at Robinson College, University of Cambridge, Cambridge, U.K., 1–4 September 2015.

References

References
1
Jelsema
C.L.
Morre
D.J.
Distribution of phospholipid biosynthetic enzymes among cell components of rat liver
J. Biol. Chem.
1978
, vol. 
253
 (pg. 
7960
-
7971
)
[PubMed]
2
Nakatsu
F.
Baskin
J.M.
Chung
J.
Tanner
L.B.
Shui
G.
Lee
S.Y.
Pirruccello
M.
Hao
M.
Ingolia
N.T.
Wenk
M.R.
De
C.P.
PtdIns4P synthesis by PI4KIIIalpha at the plasma membrane and its impact on plasma membrane identity
J. Cell Biol.
2012
, vol. 
199
 (pg. 
1003
-
1016
)
[PubMed]
3
Michell
R.H.
Inositol phospholipids in cell surface receptor function
Biochim. Biophys. Acta
1975
, vol. 
415
 (pg. 
81
-
147
)
[PubMed]
4
Helmkamp
G.M.
Jr
Harvey
M.S.
Wirtz
K.W.A.
van Deenen
L.L.M.
Phospholipid exchange between membranes. Purification of bovine brain proteins that preferentially catalyze the transfer of phosphatidylinositol
J. Biol. Chem.
1974
, vol. 
249
 (pg. 
6382
-
6389
)
[PubMed]
5
Cunningham
E.
Thomas
G.M.H.
Ball
A.
Hiles
I.
Cockcroft
S.
Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2
Curr. Biol.
1995
, vol. 
5
 (pg. 
775
-
783
)
[PubMed]
6
Kauffmann-Zeh
A.
Thomas
G.M.H.
Ball
A.
Prosser
S.
Cunningham
E.
Cockcroft
S.
Hsuan
J.J.
Requirement for phosphatidylinositol transfer protein in epidermal growth factor signalling
Science
1995
, vol. 
268
 (pg. 
1188
-
1190
)
[PubMed]
7
Thomas
G.M.H.
Cunningham
E.
Fensome
A.
Ball
A.
Totty
N.F.
Troung
O.
Hsuan
J.J.
Cockcroft
S.
An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signalling
Cell
1993
, vol. 
74
 (pg. 
919
-
928
)
[PubMed]
8
Yoder
M.D.
Thomas
L.M.
Tremblay
J.M.
Oliver
R.L.
Yarbrough
L.R.
Helmkamp
G.M.
Jr
Structure of a multifunctional protein. Mammalian phosphatidylinositol transfer protein complexed with phosphatidylcholine
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
9246
-
9252
)
[PubMed]
9
Tilley
S.J.
Skippen
A.
Murray-Rust
J.
Swigart
P.
Stewart
A.
Morgan
C.P.
Cockcroft
S.
McDonald
N.Q.
Structure-function analysis of human phosphatidylinositol transfer protein alpha bound to phosphatidylinositol
Structure
2004
, vol. 
12
 (pg. 
317
-
326
)
[PubMed]
10
Whatmore
J.
Wiedemann
C.
Somerharju
P.
Swigart
P.
Cockcroft
S.
Resynthesis of phosphatidylinositol in permeabilised neutrophils following phospholipase Cβ activation: Transport of the intermediate, phosphatidic acid from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport
Biochem. J.
1999
, vol. 
341
 (pg. 
435
-
444
)
[PubMed]
11
Garner
K.
Hunt
A.N.
Koster
G.
Somerharju
P.
Grover
E.
Li
M.
Raghu
P.
Holic
R.
Cockcroft
S.
Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) binds and transfers phosphatidic acid
J. Biol. Chem.
2012
, vol. 
287
 (pg. 
32263
-
32276
)
[PubMed]
12
Connerth
M.
Tatsuta
T.
Haag
M.
Klecker
T.
Westermann
B.
Langer
T.
Intramitochondrial transport of phosphatidic acid in yeast by a lipid transfer protein
Science
2012
, vol. 
338
 (pg. 
815
-
818
)
[PubMed]
13
Yu
F.
He
F.
Yao
H.
Wang
C.
Wang
J.
Li
J.
Qi
X.
Xue
H.
Ding
J.
Zhang
P.
Structural basis of intramitochondrial phosphatidic acid transport mediated by Ups1-Mdm35 complex
EMBO Rep.
2015
, vol. 
16
 (pg. 
813
-
823
)
[PubMed]
14
Miliara
X.
Garnett
J.A.
Tatsuta
T.
Ali
F.A.
Baldie
H.
Perez-Dorado
I.
Simpson
P.
Yague
E.
Langer
T.
Matthews
S.
Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes
EMBO Rep.
2015
, vol. 
16
 (pg. 
824
-
835
)
[PubMed]
15
Harris
W.A.
Stark
W.S.
Hereditary retinal degeneration in Drosophila melanogaster. A mutant defect associated with the phototransduction process
J. Gen. Physiol.
1977
, vol. 
69
 (pg. 
261
-
291
)
[PubMed]
16
Klinkenberg
D.
Long
K.R.
Shome
K.
Watkins
S.C.
Aridor
M.
A cascade of ER exit site assembly that is regulated by p125A and lipid signals
J. Cell Sci.
2014
, vol. 
127
 (pg. 
1765
-
1778
)
[PubMed]
17
Inoue
H.
Baba
T.
Sato
S.
Ohtsuki
R.
Takemori
A.
Watanabe
T.
Tagaya
M.
Tani
K.
Roles of SAM and DDHD domains in mammalian intracellular phospholipase A1 KIAA0725p
Biochim. Biophys. Acta
2012
, vol. 
1823
 (pg. 
930
-
939
)
[PubMed]
18
Kim
S.
Kedan
A.
Marom
M.
Gavert
N.
Keinan
O.
Selitrennik
M.
Laufman
O.
Lev
S.
The phosphatidylinositol-transfer protein Nir2 binds phosphatidic acid and positively regulates phosphoinositide signalling
EMBO Rep.
2013
, vol. 
14
 (pg. 
891
-
899
)
[PubMed]
19
Lu
C.
Vihtelic
T.S.
Hyde
D.R.
Li
T.
A neuronal-specific mammalian homolog of the Drosophila retinal degeneration B gene with expression to the retina and dentate gyrus
J. Neurosci.
1999
, vol. 
19
 (pg. 
7317
-
7325
)
[PubMed]
20
Litvak
V.
Shaul
Y.D.
Shulewitz
M.
Amarilio
R.
Carmon
S.
Lev
S.
Targeting of Nir2 to lipid droplets is regulated by a specific threonine residue within its PI-transfer domain
Curr. Biol.
2002
, vol. 
12
 (pg. 
1513
-
1518
)
[PubMed]
21
Yadav
S.
Garner
K.
Georgiev
P.
Li
M.
Espinosa
E.G.
Panda
A.
Mathre
S.
Okkenhaug
H.
Cockcroft
S.
Raghu
P.
RDGBα, a PI-PA transfer protein regulates G-protein coupled PtdIns(4,5)P2 signalling during Drosophila phototransduction
J. Cell Sci.
2015
, vol. 
128
 (pg. 
3330
-
3344
)
[PubMed]
22
Cockcroft
S.
Garner
K.
14-3-3 protein and ATRAP bind to the soluble class IIB phosphatidylinositol transfer protein RdgBbeta at distinct sites
Biochem. Soc. Trans.
2012
, vol. 
40
 (pg. 
451
-
456
)
[PubMed]
23
Garner
K.
Li
M.
Ugwuanya
N.
Cockcroft
S.
The phosphatidylinositol transfer protein, RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid binding domain interacts with the integral membrane protein, ATRAP (angiotensin II type I receptor-associated protein)
Biochem. J.
2011
, vol. 
439
 (pg. 
97
-
111
)
[PubMed]
24
Cosker
K.E.
Shadan
S.
van Diepen
M.
Morgan
C.
Li
M.
Allen-Baume
V.
Hobbs
C.
Doherty
P.
Cockcroft
S.
Eickholt
B.J.
Regulation of PI3K signalling by the phosphatidylinositol transfer protein PITP{alpha} during axonal extension in hippocampal neurons
J. Cell Sci.
2008
, vol. 
121
 (pg. 
796
-
803
)
[PubMed]
25
Shadan
S.
Holic
R.
Carvou
N.
Ee
P.
Li
M.
Murray-Rust
J.
Cockcroft
S.
Dynamics of lipid transfer by phosphatidylinositol transfer proteins in cells
Traffic
2008
, vol. 
9
 (pg. 
1743
-
1756
)
[PubMed]
26
Carlisle
F.A.
Pearson
S.
Steel
K.P.
Lewis
M.A.
Pitpnm1 is expressed in hair cells during development but is not required for hearing
Neuroscience
2013
, vol. 
248C
 (pg. 
620
-
625
)
27
Lev
S.
Hernandez
J.
Martinez
R.
Chen
A.
Plowman
G.
Schlessinger
J.
Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein
Mol. Cell. Biol.
1999
, vol. 
19
 (pg. 
2278
-
2288
)
[PubMed]
28
Lu
C.
Peng
Y.W.
Shang
J.
Pawlyk
B.S.
Yu
F.
Li
T.
The mammalian retinal degeneration B2 gene is not required for photoreceptor function and survival
Neuroscience
2001
, vol. 
107
 (pg. 
35
-
41
)
[PubMed]
29
Walker
M.T.
Rupp
A.
Elsaesser
R.
Guler
A.D.
Sheng
W.
Weng
S.
Berson
D.M.
Hattar
S.
Montell
C.
RDGB2 required for dim light input into intrinsically photosensitive retinal ganglion cells
Mol. Biol. Cell
2015
, vol. 
26
 (pg. 
3671
-
3678
)
[PubMed]
30
Vihtelic
T.S.
Goebl
M.
Milligan
S.
O'Tousa
S.E.
Hyde
D.R.
Localization of Drosophila retinal degeneration B, a membrane- associated phosphatidylinositol transfer protein
J. Cell Biol.
1993
, vol. 
122
 (pg. 
1013
-
1022
)
[PubMed]
31
Iwata
R.
Oda
S.
Kunitomo
H.
Iino
Y.
Roles for class IIA phosphatidylinositol transfer protein in neurotransmission and behavioural plasticity at the sensory neuron synapses of Caenorhabditis elegans
Proc. Natl. Acad. Sci. U.S.A.
2011
, vol. 
108
 (pg. 
7589
-
7594
)
[PubMed]
32
White
J.G.
Southgate
E.
Thomson
J.N.
Brenner
S.
The structure of the nervous system of the nematode Caenorhabditis elegans
Philos. Trans. R. Soc. Lond. B Biol. Sci.
1986
, vol. 
314
 (pg. 
1
-
340
)
[PubMed]
33
Suzuki
E.
Hirosawa
K.
Immunolocalization of a Drososphila phosphatidylinositol transfer protein (rdgB) in normal and rdgA mutant photoreceptor cells with special reference to the subrhabdomeric cisternae
J. Electron. Microsc.
1994
, vol. 
43
 (pg. 
183
-
189
)
34
Chakrabarti
P.
Kolay
S.
Yadav
S.
Kumari
K.
Nair
A.
Trivedi
D.
Raghu
P.
A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors
PLoS Genet.
2015
, vol. 
11
 pg. 
e1004948
 
[PubMed]
35
Paetkau
D.W.
Elagin
V.A.
Sendi
L.M.
Hyde
D.R.
Isolation and characterization of Drosophila retinal degeneration B suppressors
Genetics
1999
, vol. 
151
 (pg. 
713
-
724
)
[PubMed]
36
Lee
Y.J.
Shah
S.
Suzuki
E.
Zars
T.
O'Day
P.M.
Hyde
D.R.
The Drosophila dgq gene encodes a G alpha protein that mediates phototransduction
Neuron
1994
, vol. 
13
 (pg. 
1143
-
1157
)
[PubMed]
37
Wang
T.
Montell
C.
A phosphoinositide synthase required for a sustained light response
J. Neurosci.
2006
, vol. 
26
 (pg. 
12816
-
12825
)
[PubMed]
38
Kunitomo
H.
Sato
H.
Iwata
R.
Satoh
Y.
Ohno
H.
Yamada
K.
Iino
Y.
Concentration memory-dependent synaptic plasticity of a taste circuit regulates salt concentration chemotaxis in Caenorhabditis elegans
Nat. Commun.
2013
, vol. 
4
 pg. 
2210
 
[PubMed]
39
Lackner
M.R.
Nurrish
S.J.
Kaplan
J.M.
Facilitation of synaptic transmission by EGL-30 Gqalpha and EGL-8 PLCbeta: DAG binding to UNC-13 is required to stimulate acetylcholine release
Neuron
1999
, vol. 
24
 (pg. 
335
-
346
)
[PubMed]
40
Chang
C.L.
Liou
J.
Phosphatidylinositol 4,5-bisphosphate homeostasis regulated by Nir2 and Nir3 at endoplasmic reticulum-plasma membrane junctions
J. Biol. Chem.
2015
, vol. 
290
 (pg. 
14289
-
14301
)
[PubMed]
41
Chang
C.L.
Hsieh
T.S.
Yang
T.T.
Rothberg
K.G.
Azizoglu
D.B.
Volk
E.
Liao
J.C.
Liou
J.
Feedback regulation of receptor-induced Ca2+ signaling mediated by E-Syt1 and Nir2 at endoplasmic reticulum-plasma membrane junctions
Cell Rep.
2013
, vol. 
5
 (pg. 
813
-
825
)
[PubMed]
42
Kim
Y.J.
Guzman-Hernandez
M.L.
Wisniewski
E.
Balla
T.
Phosphatidylinositol-phosphatidic acid exchange by Nir2 at ER-PM contact sites maintains phosphoinositide signaling competence
Dev. Cell
2015
, vol. 
33
 (pg. 
549
-
561
)
[PubMed]
43
Keinan
O.
Kedan
A.
Gavert
N.
Selitrennik
M.
Kim
S.
Karn
T.
Becker
S.
Lev
S.
The lipid-transfer protein Nir2 enhances epithelial-mesenchymal transition and facilitates breast cancer metastasis
J. Cell Sci.
2014
, vol. 
127
 (pg. 
4740
-
4749
)
[PubMed]
44
Giordano
F.
Saheki
Y.
Idevall-Hagren
O.
Colombo
S.F.
Pirruccello
M.
Milosevic
I.
Gracheva
E.O.
Bagriantsev
S.N.
Borgese
N.
De Camilli
P.
PI(4,5)P2-dependent and Ca(2+)-regulated ER-PM interactions mediated by the extended synaptotagmins
Cell
2013
, vol. 
153
 (pg. 
1494
-
1509
)
[PubMed]
45
Suh
P.G.
Park
J.I.
Manzoli
L.
Cocco
L.
Peak
J.C.
Katan
M.
Fukami
K.
Kataoka
T.
Yun
S.
Ryu
S.H.
Multiple roles of phosphoinositide-specific phospholipase C isozymes
BMB. Rep.
2008
, vol. 
41
 (pg. 
415
-
434
)
[PubMed]
46
Michell
R.H.
Jafferji
S.S.
Jones
L.M.
Receptor occupancy dose-response curve suggests that phosphatidylinositol breakdown may be intrinsic to the mechanism of the muscarinic cholinergic receptor
FEBS Lett.
1976
, vol. 
69
 (pg. 
1
-
5
)
[PubMed]
47
Falkenburger
B.H.
Dickson
E.J.
Hille
B.
Quantitative properties and receptor reserve of the DAG and PKC branch of Gq-coupled receptor signaling
J. Gen. Physiol.
2013
, vol. 
141
 (pg. 
537
-
555
)
[PubMed]
48
Dickson
E.J.
Falkenburger
B.H.
Hille
B.
Quantitative properties and receptor reserve of the IP3 and calcium branch of Gq-coupled receptor signaling
J. Gen. Physiol.
2013
, vol. 
141
 (pg. 
521
-
535
)
[PubMed]
49
van
R.J.
Song
X.
van
R.W.
Cammer
M.
Chen
X.
Desmarais
V.
Yip
S.C.
Backer
J.M.
Eddy
R.J.
Condeelis
J.S.
EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells
J. Cell Biol.
2007
, vol. 
179
 (pg. 
1247
-
1259
)
[PubMed]
50
Loewen
C.J.
Roy
A.
Levine
T.P.
A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP
EMBO J.
2003
, vol. 
22
 (pg. 
2025
-
2035
)
[PubMed]