Phosphorylation of Thr654 but not Thr669 within the juxtamembrane domain of the EGF receptor inhibits calmodulin binding

Calcium-calmodulin (CaM) binding to the epidermal growth factor receptor (EGFR) has been shown to both inhibit and stimulate receptor activity. CaM binds to the intracellular juxtamembrane (JM) domain (Met645-Phe688) of EGFR. Protein kinase C (PKC) mediated phosphorylation of Thr654 occurs within this domain. CaM binding to the JM domain inhibits PKC phosphorylation and conversely PKC mediated phosphorylation of Thr654 or Glu substitution of Thr654 inhibits CaM binding. A second threonine residue (Thr669) within the JM domain is phosphorylated by the mitogen-activated protein kinase (MAPK). Previous results have shown that CaM interferes with EGFR-induced MAPK activation. If and how phosphorylation of Thr669 affects CaM-EGFR interaction is however not known.In the present study we have used surface plasmon resonance (BIAcore) to study the influence of Thr669 phosphorylation on real time interactions between the intracellular juxtamembrane (JM) domain of EGFR and CaM. The EGFR-JM was expressed as GST fusion proteins in Escherichia coli and phosphorylation was mimicked by generating Glu substitutions of either Thr654 or Thr669. Purified proteins were coupled to immobilized anti-GST antibodies at the sensor surface and increasing concentration of CaM was applied. When mutating Thr654 to Glu654 no specific CaM binding could be detected. However, neither single substitutions of Thr669 (Gly669 or Glu669) nor double mutants Gly654/Gly669 or Gly654/Glu669 influenced the binding of CaM to the EGFR-JM. This clearly shows that PKC may regulate EGF-mediated CaM signalling through phosphorylation of Thr654 whereas phosphorylation of Thr669 seems to play a CaM independent regulatory role. The role of both residues in the EGFR-calmodulin interaction was also studied in silico. Our modelling work supports a scenario where Thr654 from the JM domain interacts with Glu120 in the calmodulin molecule. Phosphorylation of Thr654 or Glu654 substitution creates a repulsive electrostatic force that would diminish CaM binding to the JM domain. These results are in line with the Biacore experiments showing a weak binding of the CaM to the JM domain with Thr654 mutated to Glu. Furthermore, these results provide a hypothesis to how CaM binding to EGFR might both positively and negatively interfere with EGFR-activity.     

Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC

We have recently shown that calmodulin (CaM) regulates the trafficking of epidermal growth factor receptor (EGFR) as well as the mitogen-activated protein kinase (MAPK) signalling pathway. However, the overall regulation of the MAPK pathway is achieved through a complex interplay of other several upstream effectors including G-proteins, EGF, EGFR, protein kinase C (PKC), phosphatidylinositol-3-kinase and CaM. In order to understand the role of CaM in the PKC-mediated transactivation of EGFR we have analysed the effect of a CaM antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, on the 12-O-tetradecanoylphorbol-13-acetate-mediated activation of EGFR and the subsequent MAPK activation. The results show that CaM interferes with MAPK activation and the transactivation of EGFR mediated by PKC.     

Calmodulin Modulates Mitogen-Activated Protein Kinase Activation in Response to Membrane Depolarization in PC12 Cells

Abstract: In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca²⁺ influx through voltage-gated Ca²⁺ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca²⁺ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K⁺ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca²⁺ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K⁺ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca²⁺ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.     

Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor

Nociceptin/orphanin FQ (N/OFQ) and its receptor system modulate a variety of biological functions and further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor. Two series of N/OFQ related analogues were synthesized to investigate the relationship of different modifications. We combined modifications including: (a) Phe⁴→(pF)Phe⁴; (b) Ala⁷, Ala¹¹→Aib⁷, Aib¹¹; (c) Leu¹⁴, Ala¹⁵→Arg¹⁴, Lys¹⁵. Compared with the first series, N-terminus of the second series was changed from Phe¹ to Nphe¹. All the analogues were amidated at C-terminus. These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. Results indicated that the compounds of the first series showed higher affinity and potency than N/OFQ (pK_i = 9.33; pEC₅₀ = 7.50). In particular, [(pF)Phe⁴, Aib⁷, Aib¹¹, Arg¹⁴, Lys¹⁵] N/OFQ-NH₂ was found to be a highly potent agonist with pK_i = 10.78 in binding studies and pEC₅₀ = 9.37 in mouse vas deferens assay. The second series all competitively antagonized the effects of N/OFQ in mouse vas deferens assay. [Nphe¹, (pF)Phe⁴, Aib⁷, Aib¹¹, Arg¹⁴, Lys¹⁵] N/OFQ-NH₂ was the best antagonist with pA₂ = 8.39 and showed high binding affinity with pK_i = 9.99. Thus modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist.     

Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind to membranes, changing the electrostatic surface potential: dual effect of W-13 on epidermal growth factor receptor activation

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.     

Interaction of calmodulin with synaptic plasma membranes isolated from sheep brain

Calmodulin binding to a membrane fraction enriched in synaptic plasma membranes of sheep brain cells was investigated with [¹²⁵I]calmodulin. Calmodulin binding to these membranes is Ca²⁺-dependent with a half maximal saturation at the pCa value of about 5.5. The binding is reduced by replacing Ca²⁺ with Mg²⁺, but it is significantly enhanced when both cations are present in the medium. Cation-dependent binding is specific and saturable with an apparent K_D of about 47–50 nM and a maximal capacity of about 4 pmol mg⁻¹ protein. The results indicate that synaptic plasma membranes isolated from sheep brain cells interact with calmodulin in a Ca²⁺-dependent, Mg²⁺-facilitated manner.     

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.     

Structure-activity relationships of trout bradykinin ([Arg(0),Trp(5),Leu(8)]-bradykinin)

Trout bradykinin ([Arg⁰,Trp⁵,Leu⁸]-BK) produces sustained and concentration–dependent contractions of isolated longitudinal smooth muscle from trout stomach, although mammalian BK is without effect. Circular dichroism studies have demonstrated that trout BK, unlike mammalian BK, does not adopt a stable β-turn conformation, even in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol. The myotropic actions of a series of analogs in which each amino acid in trout BK was replaced by either alanine or the corresponding D-isomer were investigated. The peptides with Ala⁴, D-Pro³, D-Trp⁵, D-Ser⁶, and D-Pro⁷ substitutions were inactive and did not act as antagonists of trout BK. The analog with [Ala⁵] was a weak partial agonist. The substitution (Arg⁰ → Ala) led to >50-fold decrease in potency but, in contrast to the importance of Phe⁸ in both BK and desArg⁹-BK in activating the mammalian B₂ and B₁ receptors respectively, substitutions at Leu⁸ in trout BK had only a minor effect on potency. Antagonists to the mammalian B₂ receptor generally contain a D-aromatic amino acid at position 7 of BK but the analog [Arg⁰,Trp⁵,D-Phe⁷,Leu⁸]-BK was a weak agonist at the trout receptor. Similarly, the potent nonpeptide mammalian B₂ receptor antagonist FR173657 was without effect on the action of trout BK. These data suggest the hypothesis that the receptor binding conformation of trout BK is defined by the central region (residues 3–7) of the peptide but is adopted only upon interaction with the receptor. The bioactive conformation is probably stabilized by an ionic interaction between Arg⁰ in the peptide and an acidic residue in the receptor.     

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

Human immune cells express ppMCH mRNA and functional MCHR1 receptor

The ubiquitously expressed Na(+)/H(+) exchanger (NHE1) plays an important role in the regulation of the intracellular pH. Induction of NHE activity by phorbol esters and inhibition of growth factor-mediated stimulation of the NHE by protein kinase C (PKC) inhibitors suggest an implication of PKCs in the regulation of the NHE. Expression of PKC isotype-specific dominant negative and constitutively active mutants or downregulation of PKC by isotype-specific antisense oligonucleotides revealed that stimulation by epidermal growth factor (EGF) or phorbol ester of the NHE in NIH3T3 cells is a PKC(alpha)-specific effect. Elevation of cytoplasmic calcium by a Ca(2+) ionophore or thapsigargin causes a growth factor-independent stimulation of the NHE predominantly mediated by calcium/calmodulin kinase II. It is concluded that in NIH3T3 cells overexpressing the EGF receptor (EGFR6 cells), EGF requires cPKC(alpha) for the activation of the NHE, while calcium/calmodulin-dependent kinases are essential in thapsigargin induced stimulation of the NHE.     

Intensification of growth factor receptor signalling by phorbol treatment of ligand-primed cells implies a dimer-stabilizing effect of protein kinase C-dependent juxtamembrane domain phosphorylation.

Protein kinase C (PKC) phosphorylates the juxtamembrane domain of many growth factor receptors, but the physiologic effect of this modification on ligand signalling and desensitisation is unclear. Here we show that PKC-dependent transmodulation of EGFR and ErbB2 signalling is schedule-specific: prolonged pre-treatment of A431 cells with the PKC agonist phorbol dibutyrate potently inhibits subsequent ligand-induced EGFR signalling as expected, but EGF pre-treatment reverses the inhibitory effect of phorbol. The agonist activity of PKC on receptor signalling is even more apparent when cells are treated with phorbol in the presence of a tyrosine phosphatase inhibitor. Because these findings suggested a synergistic interaction between tyrosine- and PKC-dependent phosphorylation events, we sought to define the interactions of tyrosine-phosphorylated and PKC-modified ErbB2 subsets within EGF-inducible hetero-oligomers. Growth factor-dependent PKC transphosphorylation takes place exclusively within endocytosed tyrosine-phosphorylated receptor oligomers. Moreover, phorbol differentially affects two ErbB2 C-terminal autophosphorylation sites: whereas phosphorylation of Tyr1222 is reduced, phosphorylation of Tyr1139 is increased. These results suggest that PKC-dependent phosphorylation of the juxtamembrane domain may contribute positively to both internalisation and signalling of ligand-activated receptors, simultaneously accelerating termination of growth factor action. We propose that transient PKC-dependent signal amplification results from enhanced stability of liganded receptor oligomers due to phosphorylation-dependent juxtamembrane domain interactions, analogous to the protein-protein binding now known to be induced by serine-threonine phosphorylation of CREB and SMAD.     

EGFR Juxtamembrane Domain, Membranes, and Calmodulin: Kinetics of Their Interaction

Calcium/calmodulin (Ca/CaM) binds to the intracellular juxtamembrane domain (JMD) of the epidermal growth factor receptor (EGFR). The basic JMD also binds to acidic lipids in the inner leaflet of the plasma membrane, and this interaction may contribute an extra level of autoinhibition to the receptor. Binding of a ligand to the EGFR produces a rapid increase in intracellular calcium, [Ca²⁺]_i, and thus Ca/CaM. How does Ca/CaM compete with the plasma membrane for the JMD? Does Ca/CaM directly pull the JMD off the membrane or does Ca/CaM only bind to the JMD after it has dissociated spontaneously from the bilayer? To answer this question, we studied the effect of Ca/CaM on the rate of dissociation of fluorescent JMD peptides from phospholipid vesicles by making kinetic stop-flow measurements. Ca/CaM increases the rate of dissociation: an analysis of the differential equations that describe the dissociation shows that Ca/CaM must directly pull the basic JMD peptide off the membrane surface. These measurements lead to a detailed atomic-level mechanism for EGFR activation that reconciles the existence of preformed EGFR dimers/oligomers with the Kuriyan allosteric model for activation of the EGFR kinase domains.     

Decorin binds to a narrow region of the epidermal growth factor (EGF) receptor,partially overlapping but distinct from the EGF-binding epitope

Decorin, a small leucine-rich proteoglycan, is a key regulator of tumor growth by acting as an antagonist of the epidermal growth factor receptor (EGFR) tyrosine kinase. To search for cell surface receptors interacting with decorin, we generated a decorin/alkaline phosphatase chimeric protein and used it to screen a cDNA library by expression cloning. We identified two strongly reactive clones that encoded either the full-length EGFR or its ectodomain. A physiologically relevant interaction between decorin and EGFR was confirmed in the yeast two-hybrid system and further validated by experiments using EGF/EGFR interaction and transient cell transfection assays. Using a panel of deletion mutants, decorin binding was mapped to a narrow region of the EGFR within its ligand-binding L2 domain. Moreover, the central leucine-rich repeat 6 of decorin was required for interaction with the EGFR. Site-directed mutagenesis of the EGFR L2 domain showed that a cluster of residues, His(394)-Ile(402), was essential for both decorin and EGF binding. In contrast, K465, previously shown to be cross-linked to epidermal growth factor (EGF), was required for EGF but not for decorin binding. Thus, decorin binds to a discrete region of the EGFR, partially overlapping with but distinct from the EGF-binding domain. These findings could lead to the generation of protein mimetics capable of suppressing EGFR function.     

The hepatitis C virus non-structural protein NS5A alters the trafficking profile of the epidermal growth factor receptor

Hepatitis C virus (HCV) frequently establishes a persistent infection, leading to chronic liver disease. The NS5A protein has been implicated in this process as it modulates a variety of intracellular signalling pathways that control cell survival and proliferation. In particular, NS5A associates with several proteins involved in the endocytosis of the epidermal growth factor receptor (EGFR) and has been previously shown to inhibit epidermal growth factor (EGF)-stimulated activation of the Ras–Erk pathway by a mechanism that remains unclear. As EGFR signalling involves trafficking to late endosomes, we investigated whether NS5A perturbs EGFR signalling by altering receptor endocytosis. We demonstrate that NS5A partially localizes to early endosomes and, although it has no effect on EGF internalization, it colocalizes with the EGFR and alters its distribution. This redistribution correlates with a decrease in the amount of active EGF–EGFR ligand–receptor complexes present in the late endosomal signalling compartment and also results in a concomitant increase in the total levels of EGFR. These observations suggest that NS5A controls EGFR signalling by diverting the receptor away from late endosomes. This represents a novel mechanism by which a viral protein attenuates cell signalling and suggests that NS5A may perturb trafficking pathways to maintain an optimal environment for HCV persistence.     

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.     

p21-activated kinase 1 (PAK1) interacts with the Grb2 adapter protein to couple to growth factor signaling

A variety of intracellular signaling pathways are linked to cell surface receptor signaling through their recruitment by Src homology 2 (SH2)/SH3-containing adapter molecules. p21-activated kinase 1 (PAK1) is an effector of Rac/Cdc42 GTPases that has been implicated in the regulation of cytoskeletal dynamics, proliferation, and cell survival signaling. In this study, we describe the specific interaction of PAK1 with the Grb2 adapter protein both in vitro and in vivo. We identify the site of this interaction as the second proline-rich SH3 binding domain of PAK1. Stimulation of the epidermal growth factor receptor (EGFR) in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. A cell-permeant TAT-tagged peptide encompassing the second proline-rich SH3 binding domain of PAK1 simultaneously blocked Grb2 and activated EGFR association with PAK1, in vitro and in vivo, indicating that Grb2 mediates the interaction of PAK1 with the activated EGFR. Blockade of this interaction decreased the epidermal growth factor-induced extension of membrane lamellae. Thus Grb2 may serve as an important mechanism for linking downstream PAK signaling to various upstream pathways.     

Epidermal growth factor receptor: mechanisms of activation and signalling

The epidermal growth factor (EGF) receptor (EGFR) is one of four homologous transmembrane proteins that mediate the actions of a family of growth factors including EGF, transforming growth factor-alpha, and the neuregulins. We review the structure and function of the EGFR, from ligand binding to the initiation of intracellular signalling pathways that lead to changes in the biochemical state of the cell. The recent crystal structures of different domains from several members of the EGFR family have challenged our concepts of these processes.     

Induction of a Nerve Growth Factor-Sensitive Kinase that Phosphorylates the DNA-Binding Domain of the Orphan Nuclear Receptor NGFI-B

Abstract: Nerve growth factor (NGF) induces the synthesis and the phosphorylation of the orphan nuclear receptor NGFI-B in PC12 cells. Previous work has shown that phosphorylation, by protein kinase A, of a specific serine in the DNA-binding domain inhibits its binding to the NGFI-B response element. Also, cytoplasmic extracts from PC12 cells phosphorylate this serine, and phosphorylation is greater in extracts from cells treated with NGF. The present work describes the induction, identification, and partial purification of a kinase (termed NGFI-B kinase I) from PC12 cell extracts that catalyzes this phosphorylation. Phosphorylation of the DNA-binding domain with this purified preparation inhibits its binding to the NGFI-B response element. The kinase is rapidly activated by treatment of the cells with NGF, and the activation lasts for at least several hours. It also is activated by fibroblast growth factor and epidermal growth factor (EGF), but the activation by EGF is quite transient. The kinase requires Mg²⁺ but will use Mn²⁺. The molecular mass of the kinase is 95–100 kDa, and it is different from protein kinase A, Fos kinase, or pp90 ^rsk . Comparison with a partially purified preparation of cyclic AMP response element-binding protein kinase, however, indicates that the two are either very similar or identical.     

Evodiamine inhibits adipogenesis via the EGFR-PKCα-ERK signaling pathway

The molecular mechanism of the anti-adipogenic effect of evodiamine (which has several capsaicin-like pharmacological actions) was investigated. The evodiamine effect was not blocked by the specific TRPV1 antagonist capsazepine in 3T3-L1 preadipocytes, whereas its effect was greatly curtailed by inhibitors of protein kinase C (PKC) and epidermal growth factor receptor (EGFR). Signal analyses showed that evodiamine stimulated the phosphorylation of EGFR, PKCα, and ERK, all of which were reduced by an EGFR inhibitor. Silencing experiments of EGFR mRNA supported the involvement of these signaling molecules in the inhibitory effect of evodiamine. An unidentified mechanism whereby evodiamine inhibits adipogenesis via the EGFR-PKCα-ERK signaling pathway was revealed.