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1.
Although agonist-dependent endocytosis of G protein-coupled receptors
(GPCRs) as a means to modulate receptor signaling has been widely studied, the
constitutive endocytosis of GPCRs has received little attention. Here we show
that two prototypical class I GPCRs, the β2 adrenergic and M3 muscarinic
receptors, enter cells constitutively by clathrin-independent endocytosis and
colocalize with markers of this endosomal pathway on recycling tubular
endosomes, indicating that these receptors can subsequently recycle back to
the plasma membrane (PM). This constitutive endocytosis of these receptors was
not blocked by antagonists, indicating that receptor signaling was not
required. Interestingly, the G proteins that these receptors couple to,
Gαs and Gαq, localized together with their
receptors at the plasma membrane and on tubular recycling endosomes. Upon
agonist stimulation, Gαs and Gαq remained
associated with the PM and these endosomal membranes, whereas β2 and M3
receptors now entered cells via clathrin-dependent endocytosis. Deletion of
the third intracellular loop (i3 loop), which is thought to play a role in
agonist-dependent endocytosis of the M3 receptor, had no effect on the
constitutive internalization of the receptor. Surprisingly, with agonist, the
mutated M3 receptor still internalized and accumulated in cells but through
clathrin-independent and not clathrin-dependent endocytosis. These findings
demonstrate that GPCRs are versatile PM proteins that can utilize different
mechanisms of internalization depending upon ligand activation.G protein-coupled receptors
(GPCRs)2 belong to a
superfamily of seven transmembrane-spanning proteins that respond to a diverse
array of sensory and chemical stimuli
(1–4).
Activation of GPCRs through the binding of specific agonists induces
conformational changes that allow activation of heterotrimeric guanine
nucleotide-binding proteins (G proteins)
(5,
6). To ensure that the signals
are controlled in magnitude and duration, activated GPCRs are rapidly
desensitized through phosphorylation carried out by G protein-coupled receptor
kinases (GRKs) (7). This
facilitates β-arrestin binding and promotes receptor uncoupling from the
G protein (8,
9). In addition to its role in
GPCRs desensitization, β-arrestins promote the translocation of the
receptor to the endocytic machinery involving clathrin and adaptor protein-2
(AP-2), thereby facilitating receptor removal from the plasma membrane
(10–15).
Once internalized, some GPCRs may even continue to signal from endosomes
(16).Although GPCR internalization is generally considered to be an
agonist-dependent phenomenon, some evidence suggests that GPCRs can be
endocytosed even in the absence of agonist, a process known as constitutive
internalization
(17–20).
The role of constitutive internalization of GPCRs is not clear. One
interesting study on cannabinoid CB1 receptors in neurons has shown that
constitutive internalization from the somatodendritic and not axonal membrane
is responsible for the overall redistribution of receptors from the
somatodentritic to the axonal membrane
(17). Another study on the
melanocortin MC4 receptor raised the possibility that constitutive endocytosis
could be a consequence of the basal activity of the receptor
(18).Even less is known about the potential trafficking of the transducer of
GPCR signaling, the G protein
(21). Generally, the binding
of the agonist to the GPCR promotes the exchange of GDP on the Gα
protein for GTP and allows the dissociation of the trimeric G protein into
Gα-GTP and Gβγ dimer subunits
(5,
22). Then, the activated G
proteins target different effectors
(23,
24). G proteins are localized
primarily to the PM where they interact with GPCRs; however, it is not known
whether G proteins always remain at the PM or whether they might move into
cells along endocytic pathways. Previous work showed that Gαs
does not colocalize with β2 receptor on internal compartments after
agonist stimulation, but the cellular distribution of Gαs was
not examined (25).In general, cargo proteins at the plasma membrane (PM) enter the cell
through a variety of endocytic mechanisms that can be divided into two main
groups: clathrin-dependent endocytosis (CDE) and clathrin-independent
endocytosis (CIE). CDE is used by PM proteins such as the transferrin receptor
(TfR) that contain specific cytoplasmic sequences recognized by adaptor
proteins allowing a rapid and efficient internalization through
clathrin-coated vesicles (26,
27). In contrast, CIE is used
by PM proteins that lack adaptor protein binding sequences including cargo
proteins such as the major histocompatibility complex class I protein (MHCI),
the glycosylphosphatidylinositol-anchored protein CD59, and integrins
(28–30).
In HeLa cells CIE is independent of, and CDE dependent on, clathrin and
dynamin and thus the two different endocytic pathways are distinct and well
defined (31). After
internalization in separate vesicles, MHCI-containing vesicles from CIE and
transferrin receptor-containing vesicles from CDE subsequently fuse with the
early endosomal compartment that is associated with Rab5 and the early
endosomal antigen 1 (EEA1)
(32). TfR is recycled back out
to the PM in Rab4- and Rab11-dependent processes. In contrast, some MHCI is
trafficked on to late endosomes and lysosomes for degradation, and some is
recycled back out to the PM along tubular endosomes that lack TfR and emanate
from the juxtanuclear area. Recycling of MHCI back to the PM requires the
activity of Arf6, Rab22, and Rab11
(33,
34).In this study, we analyzed the trafficking of GPCRs and their G proteins in
the presence and absence of agonist in HeLa cells. We examined the trafficking
of two prototypical class I GPCRs: the β2 adrenergic receptor (coupled to
Gαs) and the M3 acetylcholine muscarinic receptor (coupled to
Gαq). We find that β2 and M3 receptors traffic
constitutively via CIE, and then, in the presence of agonist, they switch to
the CDE pathway. We also examined the role of the third intracellular loop of
the M3 receptor in this process. To our knowledge, this study represents the
most comprehensive analysis of constitutive trafficking of class I GPCRs and
related Gα proteins. We demonstrate that GPCRs are versatile PM cargos
that utilize different mechanisms of internalization depending upon ligand
activation. Considering the high level of homology between class I GPCRs, this
evidence could be applicable to the other members of this family. 相似文献
2.
Lingyong Li Kristoff T. Homan Sergey A. Vishnivetskiy Aashish Manglik John J. G. Tesmer Vsevolod V. Gurevich Eugenia V. Gurevich 《The Journal of biological chemistry》2015,290(17):10775-10790
G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. 相似文献
3.
Shu-Hong Huang Ling Zhao Zong-Peng Sun Xue-Zhi Li Zhao Geng Kai-Di Zhang Moses V. Chao Zhe-Yu Chen 《The Journal of biological chemistry》2009,284(22):15126-15136
Brain-derived neurotrophic factor (BDNF) signaling through its receptor,
TrkB, modulates survival, differentiation, and synaptic activity of neurons.
Both full-length TrkB (TrkB-FL) and its isoform T1 (TrkB.T1) receptors are
expressed in neurons; however, whether they follow the same endocytic pathway
after BDNF treatment is not known. In this study we report that TrkB-FL and
TrkB.T1 receptors traverse divergent endocytic pathways after binding to BDNF.
We provide evidence that in neurons TrkB.T1 receptors predominantly recycle
back to the cell surface by a “default” mechanism. However,
endocytosed TrkB-FL receptors recycle to a lesser extent in a hepatocyte
growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner which
relies on its tyrosine kinase activity. The distinct role of Hrs in promoting
recycling of internalized TrkB-FL receptors is independent of its
ubiquitin-interacting motif. Moreover, Hrs-sensitive TrkB-FL recycling plays a
role in BDNF-induced prolonged mitogen-activated protein kinase (MAPK)
activation. These observations provide evidence for differential postendocytic
sorting of TrkB-FL and TrkB.T1 receptors to alternate intracellular
pathways.Brain-derived neurotrophic factor
(BDNF)3 has been shown
to play critical roles in vertebrate nervous system development and function
(1–3).
The actions of BDNF are dictated by two classes of cell surface receptors, the
TrkB receptor and the p75 neurotrophin receptor. BDNF binding to TrkB
receptors activates several signaling cascades, including phosphatidylinositol
3-kinase, phospholipase C, and Ras/mitogen-activated protein kinase (MAPK)
pathways, that mediate growth and survival responses to BDNF
(1,
4,
5). It has been established
that upon binding neurotrophins, Trk receptors are rapidly endocytosed in a
clathrin-dependent manner (6,
7). Postendocytic sorting of
Trk receptors to diverse pathways after ligand binding has a significant
impact on the physiological responses to neurotrophins because they also
determine the strength and duration of intracellular signaling cascades
initiated by activated Trk receptors
(8). Three alternate endocytic
pathways that Trk receptors can follow are trafficking to lysosomes for
degradation, recycling back to the plasma membrane, or being retrogradely
transported
(9–13).
The degradative pathway to lysosomes is characterized by down-regulation of
the total number of receptors at the cell surface and a decreased response to
ligand. Conversely, recycling of receptors back to the plasma membrane can
lead to functional resensitization and prolongation of cell surface-specific
signaling events. A recent study has shown that recycled and re-secreted BDNF
plays an important role in mediating the maintenance of long term potentiation
in hippocampal slices, which suggests a potential role of TrkB recycling in
long term potentiation regulation
(14).Different TrkB isoforms, including the full-length TrkB (TrkB-FL) and three
truncated isoforms named TrkB.T1, TrkB.T2, and TrkB.T-Shc, exist in the
mammalian central nervous system because of alternative splicing
(15–17).
Truncated TrkB.T1 receptor lacks the kinase domain but contains short
isoform-specific cytoplasmic domain in its place
(15,
16). Many neuronal
populations, including hippocampal and cortical neurons, express both
full-length and truncated TrkB receptors
(18,
19). TrkB.T1 is expressed at
low levels in the prenatal rodent brain, but its expression increases
postnatally, ultimately exceeding the level of full-length TrkB in adulthood
(19–22).
The physiological function of the TrkB.T1 receptor remains unclear, but it may
serve as dominant-negative regulator of full-length TrkB receptors
(23–25),
may sequester ligand and limit diffusion
(26,
27), may regulate cell
morphology and dendritic growth
(28,
29), and may even autonomously
activate signaling cascades in a neurotrophin-dependent manner
(30). TrkB-FL and TrkB.T1 are
localized to both somatodendritic and axonal compartments in neurons
(31); however, little is known
about TrkB.T1 endocytic trafficking fate upon BDNF treatment.In this study we conducted an analysis of the postendocytic fates
(degradation and recycling) of TrkB-FL and TrkB.T1 receptors in PC12 cells and
neurons. We have determined that, unlike TrkB-FL, TrkB.T1 receptors recycle
more efficiently in a default pathway to plasma surface after internalization,
which is independent of hepatocyte growth factor-regulated tyrosine kinase
substrate (Hrs). Conversely, Hrs could bind with TrkB-FL in a kinase
activity-dependent manner and regulate TrkB-FL receptors postendocytic
recycling. Hrs was identified as a tyrosine-phosphorylated protein in cells
stimulated with growth factors and cytokines
(32). Hrs is expressed in the
cytoplasm of all cells and is predominantly localized to endosomes
(33). Hrs has also been
proposed to play a role in regulating cell surface receptor postendocytic
trafficking (34). These
observations provide evidence for differential postendocytic sorting to
alternate intracellular pathways between TrkB-FL and TrkB.T1 receptors after
internalization. 相似文献
4.
Congmin Li Jenny Chan Franciose Haeseleer Katsuhiko Mikoshiba Krzysztof Palczewski Mitsuhiko Ikura James B. Ames 《The Journal of biological chemistry》2009,284(4):2472-2481
Calcium-binding protein 1 (CaBP1), a neuron-specific member of the
calmodulin (CaM) superfamily, modulates Ca2+-dependent activity of
inositol 1,4,5-trisphosphate receptors (InsP3Rs). Here we present
NMR structures of CaBP1 in both Mg2+-bound and
Ca2+-bound states and their structural interaction with
InsP3Rs. CaBP1 contains four EF-hands in two separate domains. The
N-domain consists of EF1 and EF2 in a closed conformation with Mg2+
bound at EF1. The C-domain binds Ca2+ at EF3 and EF4, and exhibits
a Ca2+-induced closed to open transition like that of CaM. The
Ca2+-bound C-domain contains exposed hydrophobic residues
(Leu132, His134, Ile141, Ile144,
and Val148) that may account for selective binding to
InsP3Rs. Isothermal titration calorimetry analysis reveals a
Ca2+-induced binding of the CaBP1 C-domain to the N-terminal region
of InsP3R (residues 1-587), whereas CaM and the CaBP1 N-domain did
not show appreciable binding. CaBP1 binding to InsP3Rs requires
both the suppressor and ligand-binding core domains, but has no effect on
InsP3 binding to the receptor. We propose that CaBP1 may regulate
Ca2+-dependent activity of InsP3Rs by promoting
structural contacts between the suppressor and core domains.Calcium ion (Ca2+) in the cell functions as an important
messenger that controls neurotransmitter release, gene expression, muscle
contraction, apoptosis, and disease processes
(1). Receptor stimulation in
neurons promotes large increases in intracellular Ca2+ levels
controlled by Ca2+ release from intracellular stores through
InsP3Rs (2). The
neuronal type-1 receptor
(InsP3R1)2
is positively and negatively regulated by cytosolic Ca2+
(3-6),
important for the generation of repetitive Ca2+ transients known as
Ca2+ spikes and waves
(1). Ca2+-dependent
activation of InsP3R1 contributes to the fast rising phase of
Ca2+ signaling known as Ca2+-induced Ca2+
release (7).
Ca2+-induced inhibition of InsP3R1, triggered at higher
cytosolic Ca2+ levels, coordinates the temporal decay of
Ca2+ transients (6).
The mechanism of Ca2+-dependent regulation of InsP3Rs is
complex (8,
9), and involves direct
Ca2+ binding sites
(5,
10) as well as remote sensing
by extrinsic Ca2+-binding proteins such as CaM
(11,
12), CaBP1
(13,
14), CIB1
(15), and NCS-1
(16).Neuronal Ca2+-binding proteins (CaBP1-5
(17)) represent a new
sub-branch of the CaM superfamily
(18) that regulate various
Ca2+ channel targets. Multiple splice variants and isoforms of
CaBPs are localized in different neuronal cell types
(19-21)
and perform specialized roles in signal transduction. CaBP1, also termed
caldendrin (22), has been
shown to modulate the Ca2+-sensitive activity of InsP3Rs
(13,
14). CaBP1 also regulates
P/Q-type voltage-gated Ca2+ channels
(23), L-type channels
(24), and the transient
receptor potential channel, TRPC5
(25). CaBP4 regulates
Ca2+-dependent inhibition of L-type channels in the retina and may
be genetically linked to retinal degeneration
(26). Thus, the CaBP proteins
are receiving increased attention as a family of Ca2+ sensors that
control a variety of Ca2+ channel targets implicated in neuronal
degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in
CaM and troponin C (18)
(Fig. 1). By analogy to CaM
(27), the four EF-hands are
grouped into two domains connected by a central linker that is four residues
longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain
non-conserved amino acids within the N-terminal region that may confer target
specificity. Another distinguishing property of CaBPs is that the second
EF-hand lacks critical residues required for high affinity Ca2+
binding (17). CaBP1 binds
Ca2+ only at EF3 and EF4, whereas it binds Mg2+ at EF1
that may serve a functional role
(28). Indeed, changes in
cytosolic Mg2+ levels have been detected in cortical neurons after
treatment with neurotransmitter
(29). Other neuronal
Ca2+-binding proteins such as DREAM
(30), CIB1
(31), and NCS-1
(32) also bind Mg2+
and exhibit Mg2+-induced physiological effects. Mg2+
binding in each of these proteins helps stabilize their Ca2+-free
state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary
structural elements (α-helices and β-strands) were derived from NMR
analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted
green, red, cyan, and yellow. Residues in the 12-residue
Ca2+-binding loops are underlined and chelating residues
are highlighted bold. Non-conserved residues in the hydrophobic patch
are colored red.Despite extensive studies on CaBP1, little is known about its structure and
target binding properties, and regulation of InsP3Rs by CaBP1 is
somewhat controversial and not well understood. Here, we present the NMR
solution structures of both Mg2+-bound and Ca2+-bound
conformational states of CaBP1 and their structural interactions with
InsP3R1. These CaBP1 structures reveal important
Ca2+-induced structural changes that control its binding to
InsP3R1. Our target binding analysis demonstrates that the C-domain
of CaBP1 exhibits Ca2+-induced binding to the N-terminal cytosolic
region of InsP3R1. We propose that CaBP1 may regulate
Ca2+-dependent channel activity in InsP3Rs by promoting
a structural interaction between the N-terminal suppressor and ligand-binding
core domains that modulates Ca2+-dependent channel gating
(8,
33,
34). 相似文献
5.
The visual photoreceptor rhodopsin is a prototypical class I (rhodopsin-like) G protein-coupled receptor. Photoisomerization of the covalently bound ligand 11-cis-retinal leads to restructuring of the cytosolic face of rhodopsin. The ensuing protonation of Glu-134 in the class-conserved D(E)RY motif at the C-terminal end of transmembrane helix-3 promotes the formation of the G protein-activating state. Using transmembrane segments derived from helix-3 of bovine rhodopsin, we show that lipid protein interactions play a key role in this cytosolic “proton switch.” Infrared and fluorescence spectroscopic pKa determinations reveal that the D(E)RY motif is an autonomous functional module coupling side chain neutralization to conformation and helix positioning as evidenced by side chain to lipid headgroup Foerster resonance energy transfer. The free enthalpies of helix stabilization and hydrophobic burial of the neutral carboxyl shift the side chain pKa into the range typical of Glu-134 in photoactivated rhodopsin. The lipid-mediated coupling mechanism is independent of interhelical contacts allowing its conservation without interference with the diversity of ligand-specific interactions in class I G protein-coupled receptors.G protein-coupled receptors (GPCRs)2 are hepta-helical membrane proteins that couple a large variety of extracellular signals to cell-specific responses via activation of G proteins. In the visual photoreceptor rhodopsin, a prototypical class I GPCR (1, 2), molecular activation processes can be monitored in real time by spectroscopic assays and analyzed in the context of several crystal structures (3–8). The primary signal for rhodopsin is the 11-cis to all-trans photoisomerization of retinal covalently bound to the apoprotein opsin through a protonated Schiff base to Lys296. Current models converge toward a picture in which “microdomains” act as conformational switches that are coupled to different degrees to the primary activation process. Two activating “proton switches” have been identified (9) as follows: breakage of an intramolecular salt bridge (10) by transfer of the Schiff base proton to its counter ion Glu-113 (11) is followed by movement of helix-6 (H6) (12, 13) in the metarhodopsin IIa (MIIa) to MIIb transition. The MIIb state takes up a proton at Glu-134 (14) in the class-conserved D(E)RY motif at the C-terminal end of helix-3 (H3) leading to the MIIbH+ intermediate (15, 16), which activates transducin (Gt), the G protein of the photoreceptor cell. Glu-134 regulates the pH sensitivity of receptor signaling (17) in membranes as reviewed previously (18), and in complex with Gt the protonated state of the carboxyl group becomes stabilized (19). This charge alteration is linked to the release of an “ionic lock,” originally described for the β2-adrenergic receptor (20), which also in rhodopsin stabilizes the inactive state (16) through interactions between the cytosolic ends of H3 and H6 (21).In the absence of a lipidic bilayer, proton uptake and H6 movement become uncoupled (15). Lipidic composition affects MII formation, rhodopsin structure, and oligomerization (22–24) and differs at the rhodopsin membrane interface from the bulk lipidic phase (25). Likewise, MII formation specifically affects lipid structure (26). Although of fundamental importance for GPCR activation, the potential implication of lipid protein interactions in “proton switching” is not clear. A functional role of Glu-134 in lipid interactions has been originally derived from IR spectra where E134Q replacement abolished changes of lipid headgroup vibrations in the MIIGt complex (19). Computational approaches emphasized the “strategic” location of the D(E)RY motif (27), and the Glu-134 carboxyl pKa may critically depend on the lipid protein interface (28). However, the implications for proton switching are not evident, and the theoretical interest is contrasted by the lack of experimental data addressing the effect of the lipidic phase on side chain protonation, secondary structure, and membrane topology of the D(E)RY motif.We have studied the coupling between conformation and protonation in single transmembrane segments derived from H3 of bovine rhodopsin. We have assessed the “modular” function of the D(E)RY motif by determining parameters not evident from the crystal structures, i.e. the pKa of the conserved carboxyl, its linkage to helical structure, and the effect of protonation on side chain to lipid headgroup distance. We show that the D(E)RY motif encodes an autonomous “proton switch” controlling side chain exposure and helix formation in the low dielectric of a lipidic phase. The data ascribe a functional role to lipid protein interactions that couple the chemical potential of protons to an activity-promoting GPCR conformation in a ligand-independent manner. 相似文献
6.
Lixia Jia Mariangela Chisari Mohammad H. Maktabi Courtney Sobieski Hao Zhou Aaron M. Konopko Brent R. Martin Steven J. Mennerick Kendall J. Blumer 《The Journal of biological chemistry》2014,289(9):6249-6257
Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K+ (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders. 相似文献
7.
James N. Hislop Anastasia G. Henry Adriano Marchese Mark von Zastrow 《The Journal of biological chemistry》2009,284(29):19361-19370
Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine δ-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized δ-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor''s hydrophobic core.A fundamental cellular mechanism contributing to homeostatic regulation of receptor-mediated signal transduction involves ligand-induced endocytosis of receptors followed by proteolysis in lysosomes. The importance of such proteolytic down-regulation has been documented extensively for a number of seven-transmembrane or G protein-coupled receptors (GPCRs),3 which comprise the largest known family of signaling receptors expressed in animals, as well as for other important signaling receptors, such as the epidermal growth factor receptor tyrosine kinase (1–5).One GPCR that is well known to undergo endocytic trafficking to lysosomes is the δ-opioid peptide receptor (DOR or DOP-R) (6). Following endocytosis, DOR traffics efficiently to lysosomes in both neural and heterologous cell models (6–8), whereas many membrane proteins, including various GPCRs, recycle rapidly to the plasma membrane (9–12). Such molecular sorting of internalized receptors between divergent recycling and degradative pathways is thought to play a fundamental role in determining the functional consequences of regulated endocytosis (2, 3, 13, 14). The sorting process that directs internalized DOR to lysosomes is remarkably efficient and appears to occur rapidly (within several min) after receptor endocytosis (11). Nevertheless, biochemical mechanisms that control lysosomal trafficking and proteolysis of DOR remain poorly understood.A conserved mechanism that promotes lysosomal trafficking of a number of membrane proteins, including various signaling receptors, is mediated by covalent modification of cytoplasmic lysine residues with ubiquitin (4, 15–17). Ubiquitination was first identified as an endocytic sorting determinant in studies of vacuolar trafficking of the yeast GPCR Ste2p (18). Subsequent studies have established numerous examples of lysyl-ubiquitination being required for sorting endocytic cargo to lysosomes and have identified conserved machinery responsible for the targeting of ubiquitinated cargo to lysosomes (3, 17, 19–22).The CXCR4 chemokine receptor provides a clear example of ubiquitin-dependent lysosomal sorting of a mammalian GPCR. Ubiquitination of the carboxyl-terminal cytoplasmic domain of the CXCR4 receptor, mediated by the E3 ubiquitin ligase AIP4, is specifically required for the HRS- and VPS4-dependent trafficking of internalized receptors to lysosomes. Blocking this ubiquitination event by Lys → Arg mutation of the receptor specifically inhibits trafficking of internalized receptors to lysosomes, resulting in recycling rather than lysosomal proteolysis of receptors after ligand-induced endocytosis (23–25).Lysosomal trafficking of DOR, in contrast, is not prevented by mutation of cytoplasmic lysine residues (26) and can be regulated by ubiquitination-independent protein interaction(s) (27, 28). Nevertheless, both wild type and lysyl-mutant DORs traffic to lysosomes via a similar pathway as ubiquitin-dependent membrane cargo and require both HRS and active VPS4 to do so (29). These observations indicate that DOR engages the same core endocytic mechanism utilized by ubiquitination-directed membrane cargo but leave unresolved whether ubiquitination of DOR plays any role in this important cellular mechanism of receptor down-regulation.There is no doubt that DOR can undergo significant ubiquitination in mammalian cells, including HEK293 cells (30–32), where lysosomal trafficking of lysyl-mutant receptors was first observed (26). Ubiquitination was shown previously to promote proteolysis of DOR by proteasomes and to function in degrading misfolded receptors from the biosynthetic pathway (30, 31). A specific role of ubiquitination in promoting proteasome- but not lysosome-mediated proteolysis of DOR has been emphasized (32) and proposed to contribute to proteolytic down-regulation of receptors also from the plasma membrane (33).To our knowledge, no previous studies have determined if DOR ubiquitination plays any role in controlling receptor proteolysis mediated by lysosomes, although this represents a predominant pathway by which receptors undergo rapid down-regulation following ligand-induced endocytosis in a number of cell types, including HEK293 cells (8). In the present study, we have taken two approaches to addressing this fundamental question. First, we have investigated in greater detail the effects of lysyl-mutation on DOR ubiquitination and trafficking. Second, we have independently investigated the role of ubiquitination in controlling lysosomal proteolysis of wild type DOR. Our results clearly establish the ability of DOR to traffic efficiently to lysosomes in the absence of any detectable ubiquitination. Further, they identify a distinct and unanticipated function of AIP4-dependent ubiquitination in regulating the later proteolytic processing of receptors and show that this distinct ubiquitin-dependent regulatory mechanism operates effectively downstream of the sorting decision that commits internalized receptors for delivery to lysosomes. 相似文献
8.
Yiliang Chen Ting Cai Haojie Wang Zhichuan Li Elizabeth Loreaux Jerry B. Lingrel Zijian Xie 《The Journal of biological chemistry》2009,284(22):14881-14890
Recent studies have ascribed many non-pumping functions to the Na/K-ATPase.
We show here that graded knockdown of cellular Na/K-ATPase α1 subunit
produces a parallel decrease in both caveolin-1 and cholesterol in light
fractions of LLC-PK1 cell lysates. This observation is further substantiated
by imaging analyses, showing redistribution of cholesterol from the plasma
membrane to intracellular compartments in the knockdown cells. Moreover, this
regulation is confirmed in α1+/– mouse liver.
Functionally, the knockdown-induced redistribution appears to affect the
cholesterol sensing in the endoplasmic reticulum, because it activates the
sterol regulatory element-binding protein pathway and increases expression of
hydroxymethylglutaryl-CoA reductase and low density lipoprotein receptor in
the liver. Consistently, we detect a modest increase in hepatic cholesterol as
well as a reduction in the plasma cholesterol. Mechanistically,
α1+/– livers show increases in cellular Src and ERK
activity and redistribution of caveolin-1. Although activation of Src is not
required in Na/K-ATPase-mediated regulation of cholesterol distribution, the
interaction between the Na/K-ATPase and caveolin-1 is important for this
regulation. Taken together, our new findings demonstrate a novel function of
the Na/K-ATPase in control of the plasma membrane cholesterol distribution.
Moreover, the data also suggest that the plasma membrane
Na/K-ATPase-caveolin-1 interaction may represent an important sensing
mechanism by which the cells regulate the sterol regulatory element-binding
protein pathway.The Na/K-ATPase, also called the sodium pump, is an ion transporter that
mediates active transport of Na+ and K+ across the
plasma membrane by hydrolyzing ATP
(1,
2). The functional sodium pump
is mainly composed of α and β subunits. The α subunit is the
catalytic component of the holoenzyme; it contains both the nucleotide and the
cation binding sites (3). So
far, four isoforms of α subunit have been discovered, and each one shows
a distinct tissue distribution pattern
(4,
5). Interestingly, studies
during the past few years have uncovered many non-pumping functions of
Na/K-ATPase
(6–10).
Recently, we have demonstrated that more than half of the Na/K-ATPase may
actually perform cellular functions other than ion pumping at least in LLC-PK1
cells (11). Moreover, the
non-pumping pool of Na/K-ATPase mainly resides in caveolae and interacts with
a variety of proteins such as Src, inositol 1,4,5-trisphosphate receptor, and
caveolin-1
(12–14).
While the interaction between Na/K-ATPase and inositol 1,4,5-trisphosphate
receptor facilitates Ca2+ signaling
(13) the dynamic association
between Na/K-ATPase and Src appears to be an essential step for ouabain to
stimulate cellular kinases
(15). More recently, we report
that the interaction between the Na/K-ATPase and caveolin-1 plays an important
role for the membrane trafficking of caveolin-1. Knockdown of the Na/K-ATPase
leads to altered subcellular distribution of caveolin-1 and increases the
mobility of caveolin-1-containing vesicles
(16).Caveolin is a protein marker for caveolae
(17). Caveolae are
flask-shaped vesicular invaginations of plasma membrane and are enriched in
cholesterol, glycosphingolipids, and sphingomyelin
(18). There are three genes
and six isoforms of caveolin. Caveolin-1 is a 22-kDa protein and is expressed
in many types of cells, including epithelial and endothelial cells. In
addition to their role in biogenesis of caveolae
(19), accumulating evidence
has implicated caveolin proteins in cellular cholesterol homeostasis
(20). For instance, caveolin-1
directly binds to cholesterol in a 1:1 ratio
(21). It was also found to be
an integral member of the intracellular cholesterol trafficking machinery
between internal membranes and plasma membrane
(22,
23). The expression of
caveolin-1 appears to be under control of
SREBPs,2 the master
regulators of intracellular cholesterol level
(24). Furthermore, knockout of
caveolin-1 significantly affected cholesterol metabolism in mouse embryonic
fibroblasts and mouse peritoneal macrophages
(25). Because we found that
the Na/K-ATPase regulates cellular distribution of caveolin-1, we propose that
it may also affect intracellular cholesterol distribution and metabolism. To
test our hypothesis, we have investigated whether sodium pump α1
knockdown affects cholesterol distribution and metabolism both in
vitro and in vivo. Our results indicate that sodium pump
α1 expression level plays a role in the proper distribution of
intracellular cholesterol. Down-regulation of sodium pump α1 not only
redistributes cholesterol between the plasma membrane and cytosolic
compartments, but also alters cholesterol metabolism in mice. 相似文献
9.
G蛋白偶联受体(G protein-coupled receptors,GPCRs)是一类重要的细胞膜表面跨膜蛋白受体超家族,具有7个跨膜螺旋结构。GPCRs的细胞内信号由G蛋白介导,可将激素、神经递质、药物、趋化因子等多种物理和化学的细胞外刺激穿过细胞膜转导到细胞内不同的效应分子,激活相应的信号级联系统进而影响恶性肿瘤的生长迁移过程。虽然目前药物市场上有很多治疗癌症的小分子药物属于G蛋白受体相关药物,但所作用的靶点集中于少数特定G蛋白偶联受体。因此,新的具有成药性的G蛋白偶联受体的开发具有很大的研究价值和市场潜力。本文主要以在癌症发生、发展中起重要作用的溶血磷脂酸(LPA),G蛋白偶联受体30(GPR30)、内皮素A受体(ETAR)等不同G蛋白偶联受体为分类依据,综述其与相关的信号通路在癌症进程中的作用,并对相应的小分子药物的临床应用和研究进展进行展望。 相似文献
10.
Qiao-Xia Hu Jun-Hong Dong Hai-Bo Du Dao-Lai Zhang Hong-Ze Ren Ming-Liang Ma Yuan Cai Tong-Chao Zhao Xiao-Lei Yin Xiao Yu Tian Xue Zhi-Gang Xu Jin-Peng Sun 《The Journal of biological chemistry》2014,289(35):24215-24225
The very large G protein-coupled receptor 1 (VLGR1) is a core component in inner ear hair cell development. Mutations in the vlgr1 gene cause Usher syndrome, the symptoms of which include congenital hearing loss and progressive retinitis pigmentosa. However, the mechanism of VLGR1-regulated intracellular signaling and its role in Usher syndrome remain elusive. Here, we show that VLGR1 is processed into two fragments after autocleavage at the G protein-coupled receptor proteolytic site. The cleaved VLGR1 β-subunit constitutively inhibited adenylate cyclase (AC) activity through Gαi coupling. Co-expression of the Gαiq chimera with the VLGR1 β-subunit changed its activity to the phospholipase C/nuclear factor of activated T cells signaling pathway, which demonstrates the Gαi protein coupling specificity of this subunit. An R6002A mutation in intracellular loop 2 of VLGR1 abolished Gαi coupling, but the pathogenic VLGR1 Y6236fsx1 mutant showed increased AC inhibition. Furthermore, overexpression of another Usher syndrome protein, PDZD7, decreased the AC inhibition of the VLGR1 β-subunit but showed no effect on the VLGR1 Y6236fsx1 mutant. Taken together, we identified an independent Gαi signaling pathway of the VLGR1 β-subunit and its regulatory mechanisms that may have a role in the development of Usher syndrome. 相似文献
11.
Rohini Shrivastava Darius K?ster Sheetal Kalme Satyajit Mayor Muniasamy Neerathilingam 《PloS one》2015,10(4)
Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well. 相似文献
12.
13.
Formin-homology (FH) 2 domains from formin proteins associate processively
with the barbed ends of actin filaments through many rounds of actin subunit
addition before dissociating completely. Interaction of the actin
monomer-binding protein profilin with the FH1 domain speeds processive barbed
end elongation by FH2 domains. In this study, we examined the energetic
requirements for fast processive elongation. In contrast to previous
proposals, direct microscopic observations of single molecules of the formin
Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed
that profilin is not required for formin-mediated processive elongation of
growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin
release the γ-phosphate of ATP on average >2.5 min after becoming
incorporated into filaments. Therefore, the release of γ-phosphate from
actin does not drive processive elongation. We compared experimentally
observed rates of processive elongation by a number of different FH2 domains
to kinetic computer simulations and found that actin subunit addition alone
likely provides the energy for fast processive elongation of filaments
mediated by FH1FH2-formin and profilin. We also studied the role of FH2
structure in processive elongation. We found that the flexible linker joining
the two halves of the FH2 dimer has a strong influence on dissociation of
formins from barbed ends but only a weak effect on elongation rates. Because
formins are most vulnerable to dissociation during translocation along the
growing barbed end, we propose that the flexible linker influences the
lifetime of this translocative state.Formins are multidomain proteins that assemble unbranched actin filament
structures for diverse processes in eukaryotic cells (reviewed in Ref.
1). Formins stimulate
nucleation of actin filaments and, in the presence of the actin
monomer-binding protein profilin, speed elongation of the barbed ends of
filaments
(2-6).
The ability of formins to influence elongation depends on the ability of
single formin molecules to remain bound to a growing barbed end through
multiple rounds of actin subunit addition
(7,
8). To stay associated during
subunit addition, a formin molecule must translocate processively on the
barbed end as each actin subunit is added
(1,
9-12).
This processive elongation of a barbed end by a formin is terminated when the
formin dissociates stochastically from the growing end during translocation
(4,
10).The formin-homology
(FH)2 1 and
2 domains are the best conserved domains of formin proteins
(2,
13,
14). The FH2 domain is the
signature domain of formins, and in many cases, is sufficient for both
nucleation and processive elongation of barbed ends
(2-4,
7,
15). Head-to-tail homodimers
of FH2 domains (12,
16) encircle the barbed ends
of actin filaments (9). In
vitro, association of barbed ends with FH2 domains slows elongation by
limiting addition of free actin monomers. This “gating” behavior
is usually explained by a rapid equilibrium of the FH2-associated end between
an open state competent for actin monomer association and a closed state that
blocks monomer binding (4,
9,
17).Proline-rich FH1 domains located N-terminal to FH2 domains are required for
profilin to stimulate formin-mediated elongation. Individual tracks of
polyproline in FH1 domains bind 1:1 complexes of profilin-actin and transfer
the actin directly to the FH2-associated barbed end to increase processive
elongation rates
(4-6,
8,
10,
17).Rates of elongation and dissociation from growing barbed ends differ widely
for FH1FH2 fragments from different formin homologs
(4). We understand few aspects
of FH1FH2 domains that influence gating, elongation or dissociation. In this
study, we examined the source of energy for formin-mediated processive
elongation, and the influence of FH2 structure on elongation and dissociation
from growing ends. In contrast to previous proposals
(6,
18), we found that fast
processive elongation mediated by FH1FH2-formins is not driven by energy from
the release of the γ-phosphate from ATP-actin filaments. Instead, the
data show that the binding of an actin subunit to the barbed end provides the
energy for processive elongation. We found that in similar polymerizing
conditions, different natural FH2 domains dissociate from growing barbed ends
at substantially different rates. We further observed that the length of the
flexible linker between the subunits of a FH2 dimer influences dissociation
much more than elongation. 相似文献
14.
A Gene Encoding Proline Dehydrogenase Is Not Only Induced by
Proline and Hypoosmolarity, but Is Also Developmentally Regulated in
the Reproductive
Organs of Arabidopsis 总被引:8,自引:0,他引:8 下载免费PDF全文
Kazuo Nakashima Rie Satoh Tomohiro Kiyosue Kazuko Yamaguchi-Shinozaki Kazuo Shinozaki 《Plant physiology》1998,118(4):1233-1241
15.
Baby G. Tholanikunnel Kusumam Joseph Karthikeyan Kandasamy Aleksander Baldys John R. Raymond Louis M. Luttrell Paul J. McDermott Daniel J. Fernandes 《The Journal of biological chemistry》2010,285(44):33816-33825
β2-Adrenergic receptors (β2-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β2-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β2-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β2-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β2-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β2-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals. 相似文献
16.
17.
Regulation of Epidermal Growth Factor Receptor Signaling by
Endocytosis and Intracellular Trafficking 总被引:13,自引:0,他引:13 下载免费PDF全文
Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction. 相似文献
18.
19.
20.
James Sinnett-Smith Rodrigo Jacamo Robert Kui YunZu M. Wang Steven H. Young Osvaldo Rey Richard T. Waldron Enrique Rozengurt 《The Journal of biological chemistry》2009,284(20):13434-13445
Rapid protein kinase D (PKD) activation and phosphorylation via protein
kinase C (PKC) have been extensively documented in many cell types cells
stimulated by multiple stimuli. In contrast, little is known about the role
and mechanism(s) of a recently identified sustained phase of PKD activation in
response to G protein-coupled receptor agonists. To elucidate the role of
biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in
these cells potently enhanced duration of ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. Cell treatment with the
preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD
activation induced by bombesin stimulation for <15 min but did not prevent
PKD catalytic activation induced by bombesin stimulation for longer times
(>60 min). The existence of sequential PKC-dependent and PKC-independent
PKD activation was demonstrated in 3T3 cells stimulated with various
concentrations of bombesin (0.3–10 nm) or with vasopressin, a
different Gq-coupled receptor agonist. To gain insight into the
mechanisms involved, we determined the phosphorylation state of the activation
loop residues Ser744 and Ser748. Transphosphorylation
targeted Ser744, whereas autophosphorylation was the predominant
mechanism for Ser748 in cells stimulated with Gq-coupled
receptor agonists. We next determined which phase of PKD activation is
responsible for promoting enhanced ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. We show, for the first
time, that the PKC-independent phase of PKD activation mediates prolonged ERK
signaling and progression to DNA synthesis in response to bombesin or
vasopressin through a pathway that requires epidermal growth factor
receptor-tyrosine kinase activity. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation
requires the identification of the molecular pathways that govern the
transition of quiescent cells into the S phase of the cell cycle. In this
context the activation and phosphorylation of protein kinase D
(PKD),4 the founding
member of a new protein kinase family within the
Ca2+/calmodulin-dependent protein kinase (CAMK) group and separate
from the previously identified PKCs (for review, see Ref.
1), are attracting intense
attention. In unstimulated cells, PKD is in a state of low catalytic (kinase)
activity maintained by autoinhibition mediated by the N-terminal domain, a
region containing a repeat of cysteinerich zinc finger-like motifs and a
pleckstrin homology (PH) domain
(1–4).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(5–7).
In response to cellular stimuli
(1), including phorbol esters,
growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR)
agonists (6,
8–16)
that signal through Gq, G12, Gi, and Rho
(11,
15–19),
PKD is converted into a form with high catalytic activity, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(5,
20).During these studies multiple lines of evidence indicated that PKC activity
is necessary for rapid PKD activation within intact cells. For example, rapid
PKD activation was selectively and potently blocked by cell treatment with
preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do
not directly inhibit PKD catalytic activity
(5,
20), implying that PKD
activation in intact cells is mediated directly or indirectly through PKCs.
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
induced by multiple GPCR agonists and other receptor ligands in a range of
cell types (for review, see Ref.
1). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation
(1,
4,
7,
17,
21). Collectively, these
findings demonstrated the existence of a rapidly activated PKC-PKD protein
kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD
activation was followed by a late, PKC-independent phase of catalytic
activation and phosphorylation induced by stimulation of the bombesin
Gq-coupled receptor ectopically expressed in COS-7 cells
(22). This study raised the
possibility that PKD mediates rapid biological responses downstream of PKCs,
whereas, in striking contrast, PKD could mediate long term responses through
PKC-independent pathways. Despite its potential importance for defining the
role of PKC and PKD in signal transduction, this hypothesis has not been
tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in
several cellular processes and activities, including signal transduction
(14,
23–25),
chromatin organization (26),
Golgi function (27,
28), gene expression
(29–31),
immune regulation (26), and
cell survival, adhesion, motility, differentiation, DNA synthesis, and
proliferation (for review, see Ref.
1). In Swiss 3T3 fibroblasts, a
cell line used extensively as a model system to elucidate mechanisms of
mitogenic signaling
(32–34),
PKD expression potently enhances ERK activation, DNA synthesis, and cell
proliferation induced by Gq-coupled receptor agonists
(8,
14). Here, we used this model
system to elucidate the role and mechanism(s) of biphasic PKD activation.
First, we show that the Gq-coupled receptor agonists bombesin and
vasopressin, in contrast to phorbol esters, specifically induce PKD activation
through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3
cells. Subsequently, we demonstrate for the first time that the
PKC-independent phase of PKD activation is responsible for promoting ERK
signaling and progression to DNA synthesis through an epidermal growth factor
receptor (EGFR)-dependent pathway. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway. 相似文献