MINIREVIEW
Submembraneous microtubule cytoskeleton: interaction of
TRPP2 with the cell cytoskeleton
Xing-Zhen Chen
1
, Qiang Li
1
, Yuliang Wu
1
, Genqing Liang
1
, Carlos J. Lara
1
and Horacio F. Cantiello
2
1 Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Canada
2 Nephrology Division, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA
Introduction
Transient receptor potential (TRP) polycystins (TRPP)
form a subfamily of the TRP superfamily of cation
channels [1]. Mutations in PKD1 or PKD2, encoding
TRPP1 (also called polycystin-1) and TRPP2 (polycys-
tin-2), respectively, account for most cases of autoso-
mal dominant polycystic kidney disease (ADPKD).
There is an increasing list of cystogenic proteins (or
called polycystic kidney disease, PKD, proteins),
including TRPP1, TRPP2, fibrocystin (PKHD1, pck
rat), polaris (Tg737, orpk mouse), inversin (inv mouse),
cystin (cpk mouse), kinesin-2 motor subunit A
(KIF3A), b-tubulin (microtubule protein), nephro-
cystin and bcl-2 [2]. There are two other TRPP mem-
bers that share 70% sequence similarity to TRPP2
and are termed TRPP3 (or polycystin-L) and TRPP5
(or polycystin-2L2) but are not likely linked to
ADPKD. TRPP members (with the exception of
TRPP1) have the typical membrane topology of TRP
channels, and share homology with domains of vol-
tage-gated cation channels, but their functions are not
as well understood as those of other TRP subfamilies,
such as TRPC and TRPV, and voltage-gated channels.
ADPKD is a prevalent genetic disorder affecting up
to 1 in 500 adults [3,4]. Autosomal recessive PKD is
less common, affecting 1 in 20 000 infants and children
[5], and is caused by mutations in the gene encoding
Keywords
actin filament; autosomal dominant
polycystic kidney disease; cellular sensor;
fibrocystin; interacting partner; ion channel;
kinesin-2; microtubule; polycystin; transient
receptor potential
Correspondence
X.-Z. Chen, Department of Physiology,
University of Alberta, 7–29 Medical
Sciences Building, Edmonton, Alberta T6G
2H7, Canada
Fax: +1 780 492 8915
Tel: +1 780 492 2294
E-mail: xzchen@ualberta.ca
(Received 15 April 2008, revised 27 May
2008, accepted 30 July 2008)
doi:10.1111/j.1742-4658.2008.06616.x
TRPP2, also called polycystin-2, the gene product of PKD2, is a membrane
protein defective in 10–15% of cases of autosomal dominant polycystic
kidney disease. Mutations in PKD2 are also associated with extrarenal dis-
orders, such as hepatic cystogenesis and cardiovascular abnormalities.
TRPP2 is a Ca-permeable nonselective cation channel present in the endo-
plasmic reticulum and plasma membrane, as well as in cilia of renal epithe-
lial and embryonic nodal cells, in which it likely forms part of a flow
sensor. Recent studies have identified a number of TRPP2-interacting pro-
teins, of which many are cytoskeletal components. Work from our and
other laboratories indicates that cytoskeletal partner proteins seem to play
important, albeit highly complex, roles in the regulation of TRPP2 expres-
sion, localization and channel function. This minireview covers current
knowledge about cytoskeletal interactions with TRPP2, and suggests that
mutations in proteins of the TRPP2-cytoskeleton complex may be impli-
cated in the pathogenesis of autosomal dominant polycystic kidney disease.
Abbreviations
ADPKD, autosomal dominant polycystic kidney disease; ER, endoplasmic reticulum; hST, human syncytiotrophoblast; IP3R, inositol 1,4,5-
triphosphate receptor; KIF3A, kinesin-2 motor subunit A; KIF3B, kinesin-2 motor subunit B; LLC-PK1, porcine kidney cells; PKD, polycystic
kidney disease; PM, plasma membrane; TRP, transient receptor potential; TRPP1, polycystin-1; TRPP2, polycystin-2; TRPP3, polycystin-L.
FEBS Journal 275 (2008) 4675–4683 ª2008 The Authors Journal compilation ª2008 FEBS 4675
fibrocystin. Renal pathogenic polycysts are fluid-filled
epithelial-lined cavities arising from various parts of
the nephron [3,6].
In addition to the kidneys, TRPP2 is also present in
several other tissues, including liver, heart, brain and
placenta. This is paralleled by liver cystogenesis and
vascular abnormalities associated with mutations in
TRPP2. Interestingly, TRPP2 is found in embryonic
nodal primary cilia where it appears to function as a
flow-dependent Ca channel, which is critical for the
development of organ left–right asymmetry. In the pri-
mary cilia of renal epithelial cells TRPP2 acts as a flow-
sensitive Ca channel, which together with TRPP1 forms
part of a tubular flow sensor [7]. TRPP3, together with
polycystin-1L3, an isoform of TRPP1, was recently
reported to be part of a sour-tasting sensor in the ton-
gue and of an acid sensor in spinal cord sensory neu-
rons [8–10]. Thus TRPP members add novel support to
the concept that TRP channels are cellular sensors. A
noteworthy element of these cases is that the roles of
partner proteins TRPP1 and polycystin-1L3 remain
elusive; it is possible that they may serve as chaperones
for trafficking, exhibit sensory function, or both.
The cytoskeletal network, comprised of actin fila-
ments, intermediate filaments and microtubules, physi-
cally connects to and ⁄or functionally regulates various
parts of the cell either directly or via a host of acces-
sory proteins. For the past few years, a number of
TRPP2 interacting partners have been discovered, of
which several are either cytoskeletal components or
cytoskeleton-associated proteins. Several studies have
shown that TRPP2 interactions with the various com-
ponents of the cytoskeleton are important for its
channel function, expression and its involvement in
sensing hydrostatic and osmotic pressure. In this mini-
review, we provide a short review of recent progress
made on the physical and functional interactions
between TRPP2 and the actin and microtubule compo-
nents of the cytoskeleton.
Channel function of TRPP2
The first demonstration of channel function of TRPP
members was in 1999 when TRPP3 was shown to
be a Ca-permeable, Ca-activated nonselective cation
channel [11]. TRPP2 was then also shown to be
a Ca-permeable nonselective cation channel, with char-
acteristics distinct from those of TRPP3 [12,13]. Native
TRPP2 was found to be present in the plasma mem-
brane (PM) and exhibits cation channel characteristics
in inner medullary collecting duct cells, Madin–Darby
canine kidney cells and Sf9 insect cells [12,14].
Endogenous TRPP2 is also present in human placenta
syncytiotrophoblast (hST) apical membranes and exhi-
bits cation selectivity and Ca-permeability upon lipid
bilayer reconstitution [12]. TRPP1 may regulate the
channel function of TRPP2. In fact, the glutathione
S-transferase-tagged TRPP1 C-terminus purified from
Escherichia coli was shown to stimulate the channel
function of in vitro translated TRPP2 reconstituted in
a lipid bilayer system [15]. This stimulatory role of
TRPP1 was confirmed by other methods in a study
using rat sympathetic neurons overexpressing the two
proteins [16]. Overexpressed TRPP2 seems more likely
to stay in the endoplasmic reticulum (ER) membrane
[13,17]. When co-expressed in Chinese hamster ovary
cells with TRPP1, TRPP2 traffics to the ER and PM
and the complex is associated with increased cell con-
ductance, likely due to PC2 channel function [17]. This
is consistent with a report that TRPP2 overexpressed
in porcine kidney cells (LLC-PK1) is primarily an ER
membrane channel involved in Ca homeostasis [18].
Interacting partners of TRPP2 and
connections to the cytoskeleton
A number of TRPP2 interacting partners have been
identified (Table 1). TRPP2 can oligomerize with itself,
TRPP1, the canonical TRP channel TRPC1, type I
inositol 1,4,5-triphosphate receptor (IP3R), epidermal
Table 1. Interacting partners of TRPP2.
Name of partner Protein type Reference
TRPP1 Membrane receptor [19,20]
EGFR Membrane receptor [23]
TRPC1 Membrane ion channel [21]
Herp ER membrane protein,
ERAD regulator
[24]
IP3R Membrane ion channel [22]
CD2AP Actin filament-associated [33]
Hax-1 Actin filament-associated [34]
Tropomyosin-1 Actin filament component [35]
a-Actinin Actin filament component [37]
Troponin-I Actin filament component [36]
KIF3A Microtubule-associated [43,44]
KIF3B Microtubule-associated [43]
mDia1 Microtubule organization
center-associated
[40]
Pericentrin Centrosome-associated [39]
PIGEA-14 Golgi- and ER-associated [38]
Id2 Helix-loop-helix [32]
PACS-1, PACS-2 Intracellular traffic sorting [31]
Phospholipase C Enzyme [23]
GSK3 Kinase [58]
P97 ATPase, ERAD regulator [24]
eIF2aTranslation initiation factor [59]
PERK Kinase, ER membrane protein [59]
TRPP2 interacts with the cytoskeleton X. -Z. Chen et al.
4676 FEBS Journal 275 (2008) 4675–4683 ª2008 The Authors Journal compilation ª2008 FEBS
growth factor receptor and Herp, an ER membrane
protein and regulator of the ER-associated degrada-
tion pathway [19–24]. The homodimerization of
TRPP2 suggests that the channel may function as a
dimer, trimer or tetramer. The TRPP2–TRPP1 channel
complex regulates cell growth and differentiation via
transcription pathways involving STAT, AP-1 or
b-catenin [25–28], couples with G proteins, and forms
a mechanical stress sensor responsive to flow [7].
TRPP1 itself directly associates with components of
the actin and intermediate filaments, such as a-actinin,
vimentin, cytokeratin and desmin [29,30]. Although the
role of TRPC1 on TRPP2 remains unknown, TRPP2
interacts with IP3R to regulate TP3R-mediated intra-
cellular Ca homeostasis [22]. This regulation seems to
be absent when their interaction is disturbed by patho-
genic mutations in TRPP2, suggesting that IP3R may
contribute to the pathogenesis of ADPKD. Phospho-
furin acidic cluster sorting (PACS-1 and PACS-2) pro-
teins interact with TRPP2 to retain it in the ER [31].
This interaction is dependent on CK2 phosphorylation
of the C-terminal residue S812 of TRPP2 and disrup-
tion in it promotes TRPP2’s trafficking to the PM.
TRPP2 associates with Id2, a member of the helix–
loop–helix protein family that is known to regulate cell
proliferation and differentiation, and this association is
regulated by TRPP1-dependent phosphorylation of
TRPP2 [32]. TRPP2 interacts with eukaryotic transla-
tion initiation factor eIF2aand ER-resident eIF2a
kinase PERK, and represses protein synthesis and cell
growth through promoting PERK-dependent phos-
phorylation of eIF2a[59].
TRPP2 associates with CD2AP, Hax-1, tropomyo-
sin-1, a-actinin and troponin-I [33–37], indicating indir-
ect and direct attachments to the cytoskeleton, and
with Golgi- and ER-associated protein PIGEA-14 [38].
TRPP2 also interacts with pericentrin at the centro-
some and with mammalian diaphanous 1 protein
(mDial) at the spindles of mitotic cells [39,40], suggest-
ing that TRPP2 at the microtubule organization center
is implicated in cell morphology and kinesis, and in
particular, in cilium growth and the function of ciliated
cells. Thus, among the physical interacting partner pro-
teins of TRPP2 identified to date about half are cyto-
skeleton or cytoskeleton-associated proteins. PKD is
associated with several abnormalities in cellular func-
tion, such as cell proliferation, differentiation and
solute transport. Interactions of these partner proteins
with TRPP2 may be implicated in these cellular func-
tions (Fig. 1). Although mutations in a particular part-
ner protein may not be sufficient for cyst formation to
occur, they may contribute to and modulate the sever-
ity of cystic disease through interactions with TRPP2.
CD2AP directly binds F-actin [33,41], whereas Hax-1
links to F-actin via cortactin [34]. Tropomyosin is an
actin-binding protein having multiple isoforms and
splicing variants in muscle as well as non-muscle cells,
including renal epithelial cells. Actin and tropomyosin
appear to act jointly to balance plasma membrane
mobility and stability. Tropomyosins are involved in
various processes, such as cell–cell adhesion, cell mor-
phogenesis and stability, and suppression of neoplastic
growth, suggesting that cyst formation might be due in
part to an altered interaction between tropomyosin
and a pathogenic TRPP2 mutant. However, it will be
interesting to examine in future studies whether, and if
so how, tropomyosin is implicated in cystogenesis as
an actin cytoskeleton component or as a neoplasticity
suppressor. In fact, because cystic epithelia show a cer-
tain similarity to neoplasticity they are sometimes
referred to as being ‘neoplastic in disguise’. Also,
whether and how tropomyosin modulates TRPP2
channel activity remains to be determined. Troponin-I
is an important actin-binding protein and represents
the inhibitory subunit of the troponin complex
(composed of troponin-I, -C and -T) involved in
Ca-dependent muscle contraction. Two additional
features of troponin-I are worthy of attention: the
presence of troponin-I in non-muscle cells and its role
as an angiogenesis inhibitor. If troponin-I is implicated
in cystogenesis, it remains to be determined whether its
property as a muscle actin cytoskeleton component or
as an angiogenesis inhibitor accounts for the implica-
tion. Interestingly, TRPP3 binds troponin-I as well
[42], despite the fact that the sequence identity between
the C-termini of TRPP2 and TRPP3 is only 42%. In
fact, yeast two-hybrid assays indicate that there are
multiple binding domains in troponin-I, but whether
these domains are the same for binding TRPP2 and
TRPP3 is still unknown. a-Actinin is an actin-binding
and actin-bundling protein important in cytoskeleton
organization, cell adhesion, proliferation and migra-
tion. Both the intracellular N- and C-termini of
Solute transport
Proliferation,
morphology
Cilium flow sensing Tubulogenesis,
differentiation
Cytoskeleton
Proliferation
α-actinin, KIF3A,
Fibrocystin+KIF3B
TRPP1
TRPP1, CD2AP,
tropomysoin,
troponin-I, Hax-1,
kinesin-2, mDial,
α-actinin,
pericentrin
TRPP2
AP-1, β-catenin
JAK/STAT
Fig. 1. Effects of interactions of TRPP2 with cytoskeleton compo-
nents or associated proteins on cellular functions. Partner proteins
are in grey boxes.
X. -Z. Chen et al. TRPP2 interacts with the cytoskeleton
FEBS Journal 275 (2008) 4675–4683 ª2008 The Authors Journal compilation ª2008 FEBS 4677
TRPP2 associate with a-actinin, as revealed by yeast
two-hybrid and in vitro binding assays [37]. TRPP2
also co-precipitates with a-actinin in HEK 293 and
Madin–Darby canine kidney cells, rat kidney and heart
tissues, and human syncytiotrophoblast apical mem-
brane vesicles. The two proteins also partially co-loca-
lize in epithelial Madin–Darby canine kidney cells and
inner medullary collecting duct cells, NIH3T3 fibro-
blasts and hST vesicles.
More recently, a microtubule-associated motor pro-
tein complex, kinesin-2, was found to interact with
both TRPP2 and fibrocystin [43,44]. It was demon-
strated that the kinesin-2 motor subunit B (KIF3B)
mediates complex formation between TRPP2 and
fibrocystin, thus serving as the first discovered molecu-
lar linker between ADPKD and autosomal recessive
polycystic kidney disease [43]. This raises questions as
to whether microtubules and kinesin-2 have functional
roles in TRPP2. Despite these discoveries of physical
associations, much remains to be determined with
respect to functional roles of the cytoskeleton-related
interacting partners of the TRPP2 channel.
Functional modulation by the actin
filament
The actin filament exerts functional effects on ion
channels, for example, actin filaments on epithelial
sodium channels [45] and cystic fibrosis transmem-
brane conductance regulator (CFTR) [46]. Recent
reports have demonstrated that a-actinin regulates the
activity of a number of channels, for example, the
muscle-type a-actinin modulates the channel function
of K channel Kv1.5, L-type Ca channel, and NMDA
receptor through direct binding [47–50]. Using com-
mercial non-muscle-type a-actinin protein with either
in vitro translated TRPP2 protein or TRPP2 enriched
from human placenta cells in a lipid bilayer system, we
found that a-actinin substantially increases TRPP2
channel activity, by increasing channel open probabil-
ity, but not single-channel conductance [37]. This sug-
gests that a-actinin binds the TRPP2 channel to
regulate its gating rather than affecting the physical
channel pore of TRPP2. Interestingly, although there
is no evidence for direct binding of actin with TRPP2,
by using G-actin, actin-filament disrupter cytochala-
sin D or actin-severing gelsolin [51], the actin filament
was found to also exert functional effects on TRPP2.
Further, these effects occur in an a-actinin-dependent
manner in the lipid bilayer system, indicating that the
action of the actin filament is mediated via intermedi-
ate, TRPP2 binding partner proteins such as a-actinin.
Of note, it is still possible that other linker proteins,
such as tropomyosin, also mediate the action of actin
on TRPP2 channel function in living cells. Thus, the
actin cytoskeleton anchors TRPP2 to the membrane
not only for structural purposes, but also regulates
channel function in a way that could be as complex as
its own structure.
Evidence supporting the actin cytoskeleton and
TRPP2 as an integral part of a sensor was provided
when using apical membrane vesicles of human hST, a
highly intricate epithelial tissue essential for the mater-
nal–fetal transfer of solutes such as ions, in lipid
bilayer reconstitution. TRPP2 was previously shown to
be abundantly expressed in hST. It was recently
reported that both hydrostatic and osmotic pressure
stimulate TRPP2 channel activity, and that stimulation
by the two physical factors was abolished when vesi-
cles were pre-treated with cytochalasin D. This indi-
cates that TRPP2 and the actin filament together, but
not TRPP2 alone, confer TRPP2 sensitivity to these
physical factors [52]. As revealed by immunofluores-
cence, cytochalasin D disrupts co-localization between
TRPP2 and the actin filament in the vicinity of the
PM. This observation leads to the concept that the
cytoskeleton, together with TRPP2, is an integral part
of the sensing element responsive to hydrostatic and
osmotic changes. Given that the cytoskeleton is in
complex with numerous other membrane proteins it
may be implicated in a variety of membrane sensing
roles.
Modulation of TRPP2 by microtubular
structures
Polycystins and several other PKD proteins are present
in renal and other epithelial cilia. A cilium is com-
posed of a microtubule, microtubule-associated motor
proteins and accessories, and a surface membrane in
which PKD and other membrane proteins are likely
inserted. Cystogenic proteins are very different in their
sequence and topology but, following pathogenic
mutations, are associated with significant phenotype
similarity. One important common thread is that the
majority of cystogenic proteins are present in renal
epithelial primary cilia. Thus, defects in cilia structure
or function, or defects in the interaction between cili-
ary membrane proteins and microtubule structures,
may be associated with cystogenesis. Another impor-
tant common thread is that cystogenic proteins directly
or indirectly associate with the cytoskeleton network,
in particular, the microtubule. It is interesting to note
that cystogenic proteins tubulin and kinesin-2 are both
microtubule-associated proteins. Although mutations
in components of the microtubule may not necessarily
TRPP2 interacts with the cytoskeleton X. -Z. Chen et al.
4678 FEBS Journal 275 (2008) 4675–4683 ª2008 The Authors Journal compilation ª2008 FEBS
be cystogenic, altered interactions between these
components and PKD proteins may be factors that
contribute to or modulate cyst progression (Fig. 1).
It was recently revealed that TRPP2 physically binds
KIF3A and KIF3B, the two motor subunits of the
kinesin-2 complex, a microtubule-associated motor
machine transporting cargo and vesicles to the plus
end of microtubules. The non-motor subunit KAP3
links kinesin-2 with various cargo proteins, including
fodrin and the tumor suppressor adenomatous polypo-
sis coli [53]. Because TRPP2 directly binds KIF3A and
KIF3B, but not KAP3, TRPP2 seems unlikely to be a
cargo protein. Thus, the main physiological role of the
TRPP2–kinesin-2 interaction does not appear to be to
transport TRPP2 to the destination by kinesin-2. In
other words, in the complexing of KIF3A and KIF3B
with TRPP2, KIF3A and KIF3B may play a ‘nontra-
ditional’ role. Interestingly, KIF3B, but not KIF3A,
also binds fibrocystin and mediates the complexing
between TRPP2 and fibrocystin. Thus, KIF3B not
only anchors the two PKD proteins, TRPP2 and fibro-
cystin, to the microtubular cytoskeleton, but also
serves as a molecular linker for dominant and recessive
PKD. Furthermore, in a reconstituted lipid bilayer
system, although KIF3B does not itself functionally
modulate TRPP2 channel, it mediates functional
stimulation of the TRPP2 channel by fibrocystin [43].
The triplex TRPP2–KIF3B–fibrocystin is presumably
present in the primary cilia of ciliated renal epithelial
cells and perinuclear regions of subconfluent cells. It
remains to be determined whether in a more physiolo-
gical model fibrocystin modulates TRPP2 channel
function in the presence of KIF3B. In contrast to
KIF3B, KIF3A does not bind fibrocystin and directly
stimulates TRPP2 channel activity. Further, stimula-
tion of the TRPP2 channel by fibrocystin via KIF3B,
or by KIF3A via direct binding [44], does not require
the hydrolysis of ATP. Taken together, this suggests
that the role of KIF3B in TRPP2–KIF3B–fibrocystin
is different from its ‘traditional role’ as a motor sub-
unit of kinesin-2. Of note, KIF3A and KIF3B were
found to be abundantly present in the nucleus (Wu Y
and Chen XZ, unpublished data), which supports the
suggestion that the two proteins have ‘nontraditional’
physiological roles. For example, it is possible that the
TRPP2–KIF3B–fibrocystin triplex is part of a sensor
in the cilium membrane. It remains unclear whether
this triplex is different from the TRPP2–TRPP1 com-
plex, or they are in the same large protein complex.
In dividing cells, TRPP2 and mDia1 were found to
be co-localized with the mitotic spindles of microtu-
bules. This TRPP2 localization is regulated by mDia1
in HeLa cells [40], suggesting a potential role of
TRPP2 in cell proliferation. Interestingly, mDia1 is
known to interact indirectly with actin filaments
through profilin [54] and to coordinate microtubules
[55]. We recently also found that TRPP2 partially
co-localizes with tubulin in the midbody of mitotic
cells at late telophase (Fig. 2). Together with the fact
that TRPP2 interacts with the microtubule-associated
motor protein kinesin-2 [43,44], these data suggest that
TRPP2 may be actively involved in kinesin-mediated
TRPP2 Acet a-tubulin DAPI Merge
Fig. 2. Localization of TRPP2 to the midbody of inner medullary collecting duct cells. TRPP2 and acetylated a-tubulin were stained using antibodies
raised from rabbit (cat # sc-25749; Santa Cruz, Santa Cruz, CA, USA) and mouse (cat # T7451; Sigma Aldrich, Oakville, Canada), respectively.
X. -Z. Chen et al. TRPP2 interacts with the cytoskeleton
FEBS Journal 275 (2008) 4675–4683 ª2008 The Authors Journal compilation ª2008 FEBS 4679