Abstract MP20: Optogenetic Control Of Pip2 To Assess Mechanisms Regulating The Epithelial Sodium Channel

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Crystal R Archer ◽  
Amanpreet Kaur ◽  
Tarek Mohamed ◽  
James D Stockand
Keyword(s):  
Binding Sites ◽  
Hek293 Cells ◽  
Proper Function ◽  
Small Decrease ◽  
Kidney Tubules ◽  
Wild Type ◽  
N Terminus ◽  
Patch Clamp Electrophysiology ◽  
G Protein Coupled

The epithelial Na + channel (ENaC) plays a key role in Na + transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na + reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP 2 binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP 2 binding sites, by controlled depletion of PIP 2 . We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP 2 levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. A fluorescent Na + indicator, was used to monitor ENaC activity by tracking the relative intracellular Na + levels. Upon optogenetic-controlled depletion of PIP 2 , Na + levels decreased in cells expressing wt ENaC. Mutations to the PIP 2 sites of ENaC were expected to have no change in Na + levels upon PIP 2 depletion due to the disruption of PIP 2 binding. As a control, mutations to non-PIP 2 binding sites were included, and were expected to have decreased Na + levels similar to wt ENaC. Interestingly, mutation of each independent PIP 2 site resulted in only a small decrease of intracellular Na + , compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP 2 binding site, resulted in a significant increase of Na + upon PIP 2 depletion. We performed patch clamp electrophysiology and found that the ENaC recordings corresponded to the Na + fluctuations. These data suggest that the residues surrounding the PIP 2 binding sites play a significant role in the affinity of PIP 2 for ENaC. The role of these other domains in PIP 2 binding is still under investigation.

scholarly journals Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

2007 ◽  
Vol 81 (15) ◽  
pp. 7833-7843 ◽  
Author(s):  
Joshua C. Grieger ◽  
Jarrod S. Johnson ◽  
Brittney Gurda-Whitaker ◽  
Mavis Agbandje-McKenna ◽  
R. Jude Samulski
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Wild Type ◽  
Dot Blot ◽  
Adeno Associated Virus ◽  
N Terminus ◽  
Localization Signal ◽  
Significant Effort

ABSTRACT Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.

Abstract 425: Protein Kinase Activity of Pi3kγ Regulates Insulin-βArs Cross-talk

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Anita SAHU ◽  
Sromona Mukherjee ◽  
Kate Stenson ◽  
Maradumane L Mohan ◽  
Sathyamangla V Prasad
Keyword(s):  
Hek293 Cells ◽  
Protein Kinase Activity ◽  
Pi3k Inhibitors ◽  
Pp2a Activity ◽  
Phosphatase 2A ◽  
Β Blocker ◽  
Sirna Knockdown ◽  
G Protein Coupled ◽  
Diabetes And Obesity

β adrenergic receptor (βAR) function is regulated by G-protein coupled receptor (GPCR) kinase (GRK) driven desensitization and protein phosphatase 2A (PP2A) mediated resensitization to its classical agonist. Surprisingly, Insulin modulates (βARs) function thus regulating cardiac function. Although insulin is known to modulate βAR function through GRKs, less is known about insulin mediated resensitization mechanisms. PI3Kγ is activated by GPCRs regulates resensitization by inhibiting PP2A activity. Therefore, we tested whether insulin could mediate βAR dysfunction through inhibition of resensitization. Co-immunoprecipitation in cardiac lysates and surface plasmon resonance (SPR) with purified proteins show that PI3Kγ interacts with GRK-2 resulting in recruitment of GRK2-PI3Kγ to the β2AR complex upon insulin. Furthermore, use of PI3K inhibitors significantly reduced Insulin-stimulated β2ARs phosphorylation in HEK293 cells. The role of PI3Kγ was further validated by the loss of insulin stimulated β 2 AR phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs), shRNA and CRIPSR knockdown of PI3Kγ. Data shows that PI3Kγ inhibits PP2A activity at the βAR complex upon insulin while loss of PI3Kγ unravels this inhibition resulting in increased PP2A activity leading to β2AR dephosphorylation and resensitization. Mechanistically, PI3Kγ inhibits PP2A activity at the β2AR complex by phosphorylating an endogenous inhibitor of PP2A (I2PP2A). CRISPR knockout and siRNA knockdown of endogenous I2PP2A in HEK293 cells restored PP2A activity resulting in β2AR dephosphorylation despite PI3Kγ. Furthermore, β blocker (propranolol) pretreatment did not affect β2AR phosphorylation and there was no β-arrestin recruitment to the βAR complex with Insulin. Together these studies show that Insulin mediates β2AR desensitization through β-agonist and β-arrestin independent mechanisms wherein, PI3Kγ-mediated regulation of PP2A activity plays a pivotal role in cardiac βAR function in hyperinsulemic conditions like diabetes and obesity.

The Role of N-Linked Glycosylation in Expression and Function of ADAMTS13

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3086-3086
Author(s):  
Agata Nowak ◽  
Chan Kwo Chion ◽  
Michael Laffan ◽  
Thomas A J McKinnon
Keyword(s):  
Enzymatic Activity ◽  
Protein Modification ◽  
Point Mutations ◽  
Proper Function ◽  
Wild Type ◽  
Rich Domain ◽  
Cellular Processing ◽  
High Mannose ◽  
Complex Glycans

Abstract N-linked glycosylation is an important co-translational protein modification affecting protein folding, secretion, stability & function. We have previously demonstrated that N-linked glycosylation of Von Willebrand Factor (VWF) modulates its interaction with ADAMTS13, the plasma metalloprotease responsible for controlling VWF multimeric size. In the present study we have investigated the role of N-linked glycosylation in modulating ADAMTS13 expression, secretion & function. Lectin analysis of plasma derived & recombinant ADAMTS13 (rAD13) demonstrated that ADAMTS13 presents sialic acid & galactose residues, indicating the presence of complex glycans structures. Analysis with the lectin GNL, specific for high mannose structures showed that plasma derived ADAMTS13 also contained a small portion of basic high mannose glycans & this was increased in rAD13. Enzymatic modification of rAD13 with PNGase F to remove whole N-linked glycans & neuraminidase to remove terminal sialic residues were completely effective. However, neither modification affected the enzymatic activity of ADAMTS13 towards VWF, indicating that following secretion ADAMTS13 N-linked glycans are expendable. Using an ADAMTS13 ELISA and western blotting, we showed that expression of rAD13 in the presence of Tunicamycin prevented N-linked glycosylation & prevented secretion, indicating that N-linked glycosylation is essential for secretion of ADAMTS13. Expression in the presence of the glucosidase inhibitor castanospermine, that inhibits binding to the endoplasmic lectin chaperones calnexin & calreticulin, also prevented secretion of ADAMTS13 suggesting that glycan dependent interaction with these chaperones is essential for the correct cellular processing of ADAMTS13 To further explore these observations, 10 individual point mutations encoding asparagine to glutamine changes were generated to prevent glycosylation at each of the 10 N-linked glycans sites in ADAMTS13: N462 & N46Q in the metalloprotease (MP) domain, N552Q in the cysteine rich domain, N579Q, N614Q & N667Q in the spacer domain, N707Q in the second Thrombospondin repeat (TSR), N828Q in the forth TSR and N1235Q and N1354Q in the CUB domains. Mutations N42Q, N579Q, N614Q, N707Q, N828Q and N1354Q caused only a slight to moderate reduction in ADAMTS13 secretion; 57%, 62%, 50%, 84%, 70% and 68% of wild type respectively. Mutations N552Q and N667Q significantly reduced ADAMTS13 secretion to 23% and 25% of wild type respectively indicating that glycosylation of the cysteine rich & spacer domains is required for efficient secretion. Significantly, mutation N46Q in the MP domain abolished secretion resulting in intracellular retention of ADAMTS13. All the secreted mutants with the exception of N828Q and N1235Q were able to effectively proteolyse the short VWF-115 substrate and full length VWF (FL-VWF). Interestingly, N828Q was demonstrated to have reduced activity towards VWF-115 and FL-VWF under both static and flow conditions. Although binding to immobilised VWF A2 domain and FL-VWF was not altered, a difference was observed in the binding of N828Q to globular FL-VWF. Conversely, mutation of N1235 increased activity of ADAMTS13 towards VWF. Together these data demonstrate that N-linked glycosylation of ADAMTS13, in particular at N46, is essential for efficient cellular processing & secretion of the enzyme. Following secretion the N-linked glycans are not required for proper function; however glycosylation of TSR 4 and the CUB domains are required for ADAMTS13 to assume a correct conformation for proper enzymatic activity towards VWF.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

scholarly journals Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice

2006 ◽  
Vol 191 (1) ◽  
pp. 197-205 ◽  
Author(s):  
Takaharu Maruyama ◽  
Kenichi Tanaka ◽  
Jun Suzuki ◽  
Hiroyuki Miyoshi ◽  
Naomoto Harada ◽  
...  
Keyword(s):  
Bile Acid ◽  
G Protein ◽  
Physiological Role ◽  
Wild Type ◽  
Fat Accumulation ◽  
Acid Receptor ◽  
Bile Acid Receptor ◽  
The Impact ◽  
G Protein Coupled

G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) is a novel G protein-coupled receptor for bile acid. Tissue distribution and cell-type specificity of Gpbar1 mRNA suggest a potential role for the receptor in the endocrine system; however, the precise physiological role of Gpbar1 still remains to be elucidated. To investigate the role of Gpbar1 in vivo, the Gpbar1 gene was disrupted in mice. In homozygous mice, total bile acid pool size was significantly decreased by 21–25% compared with that of the wild-type mice, suggesting that Gpbar1 contributes to bile acid homeostasis. In order to assess the impact of Gpbar1 deficiency in bile acid homeostasis more precisely, Gpbar1 homozygous mice were fed a high-fat diet for 2 months. As a result, female Gpbar1 homozygous mice showed significant fat accumulation with body weight gain compared with that of the wild-type mice. These findings were also observed in heterozygous mice to the same extent. Although the precise mechanism for fat accumulation in female Gpbar1 homozygous mice remains to be addressed, these data indicate that Gpbar1 is a potential new player in energy homeostasis. Thus, Gpbar1-deficient mice are useful in elucidating new physiological roles for Gpbar1.

scholarly journals Differential phosphorylation signals control endocytosis of GPR15

2017 ◽  
Vol 28 (17) ◽  
pp. 2267-2281 ◽  
Author(s):  
Yukari Okamoto ◽  
Sojin Shikano
Keyword(s):  
Surface Density ◽  
Functional Role ◽  
Control Cell ◽  
Phosphorylation Site ◽  
Hek293 Cells ◽  
C Terminus ◽  
Differential Phosphorylation ◽  
G Protein Coupled

GPR15 is an orphan G protein–coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the β-arrestin– and GPCR kinase–dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis.

scholarly journals Corepression of the P1 Addiction Operon by Phd and Doc

1998 ◽  
Vol 180 (23) ◽  
pp. 6342-6351 ◽  
Author(s):  
Roy Magnuson ◽  
Michael B. Yarmolinsky
Keyword(s):  
Fusion Protein ◽  
Binding Sites ◽  
Wild Type ◽  
Dna Complexes ◽  
Present Evidence ◽  
Cooperative Interactions ◽  
Regulatory Properties

ABSTRACT The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmid-free segregants, and Phd, an unstable antidote that neutralizes the toxin. Additionally, these products repress transcription of the operon. The antidote binds to two adjacent sites in the promoter. Here we present evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation,docH66Y. The DocH66Y protein retained the regulatory properties of the wild-type protein, but not its toxicity. In vivo, DocH66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Binding of toxin to antidote may be the physical basis for the neutralization of toxin. DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. Although DocH66Y enhanced the binding of Phd to two adjacent Phd-binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates cooperative interactions between adjacent Phd-binding sites. Several electrophoretically distinct protein-DNA complexes were observed with different amounts of DocH66Y relative to Phd. Maximal repression and specificity of DNA binding were observed with subsaturating amounts of DocH66Y relative to Phd. Analogous antidote-toxin pairs appear to have similar autoregulatory circuits. Autoregulation, by dampening fluctuations in the levels of toxin and antidote, may prevent the inappropriate activation of the toxin.

Demonstration and characterization of motilin-binding sites in the rabbit cerebellum

1997 ◽  
Vol 272 (5) ◽  
pp. G994-G999 ◽  
Author(s):  
I. Depoortere ◽  
T. L. Peeters
Keyword(s):  
Binding Sites ◽  
Molecular Layer ◽  
Physiological Role ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Lower Affinity ◽  
Erythromycin A ◽  
G Protein Coupled

This is the first report on central motilin receptors. Autoradiography on cerebellar slices revealed specific motilin-binding sites in the molecular layer of the cortex. Scatchard analysis of cold saturation studies showed the existence of a high-(pKd,hi = 9.07 +/- 0.09, where pKd is the negative logarithm of the dissociation constant) and a low-affinity binding site (pKd,lo = 6.56 +/- 0.09). Similar affinities were found with rabbit motilin and with the porcine (po) antagonist [Phe3, Leu13]po-motilin. Feline and canine motilin had a markedly lower affinity for the low-affinity site (pKd,lo = 5.29 and 4.58, respectively); chicken motilin had a lower affinity for both sites (pKd,hi = 8.36, pKd,lo = 3.97). Erythromycin A and its derivative N-trimethyl erythromycin A cnol ether also bound to cerebellar motilin receptors (pKd,hi = 7.29 and 8.91, respectively). Structure-activity studies with motilin fragments and the potency ranking of agonists suggest that a novel subtype receptor of motilin may exist in the brain. Guanosine 5'-O-(3-thiotriphosphate) (0.1 mM) reduced the number and the affinity for the high-affinity binding sites, which is evidence for G protein-coupled receptors. Our findings open new perspectives for the study of the physiological role of motilin.

scholarly journals Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-κB and AP-1

2005 ◽  
Vol 25 (14) ◽  
pp. 5933-5946 ◽  
Author(s):  
Heping Yang ◽  
Nathaniel Magilnick ◽  
Candy Lee ◽  
Denise Kalmaz ◽  
Xiaopeng Ou ◽  
...  
Keyword(s):  
Binding Sites ◽  
Promoter Activity ◽  
Family Members ◽  
Catalytic Subunit ◽  
Mrna Levels ◽  
Wild Type ◽  
Glutamate Cysteine Ligase ◽  
Responsive Element ◽  
Antioxidant Responsive Element

ABSTRACT Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tert-Butylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidant-responsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-κB family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-κB binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-κB family members.

scholarly journals Heterogeneity of signal transduction by Na-K-ATPase α-isoforms: role of Src interaction

2018 ◽  
Vol 314 (2) ◽  
pp. C202-C210 ◽  
Author(s):  
Hui Yu ◽  
Xiaoyu Cui ◽  
Jue Zhang ◽  
Joe X. Xie ◽  
Moumita Banerjee ◽  
...  
Keyword(s):  
Binding Sites ◽  
Direct Interaction ◽  
Causative Role ◽  
Wild Type ◽  
Caveolin 1 ◽  
Proposed Model ◽  
Signaling Function ◽  
Domain Interactions ◽  
Key Residues

Of the four Na-K-ATPase α-isoforms, the ubiquitous α1 Na-K-ATPase possesses both ion transport and Src-dependent signaling functions. Mechanistically, we have identified two putative pairs of domain interactions between α1 Na-K-ATPase and Src that are critical for α1 signaling function. Our subsequent report that α2 Na-K-ATPase lacks these putative Src-binding sites and fails to carry on Src-dependent signaling further supported our proposed model of direct interaction between α1 Na-K-ATPase and Src but fell short of providing evidence for a causative role. This hypothesis was specifically tested here by introducing key residues of the two putative Src-interacting domains present on α1 but not α2 sequence into the α2 polypeptide, generating stable cell lines expressing this mutant, and comparing its signaling properties to those of α2-expressing cells. The mutant α2 was fully functional as a Na-K-ATPase. In contrast to wild-type α2, the mutant gained α1-like signaling function, capable of Src interaction and regulation. Consistently, the expression of mutant α2 redistributed Src into caveolin-1-enriched fractions and allowed ouabain to activate Src-mediated signaling cascades, unlike wild-type α2 cells. Finally, mutant α2 cells exhibited a growth phenotype similar to that of the α1 cells and proliferated much faster than wild-type α2 cells. These findings reveal the structural requirements for the Na-K-ATPase to function as a Src-dependent receptor and provide strong evidence of isoform-specific Src interaction involving the identified key amino acids. The sequences surrounding the putative Src-binding sites in α2 are highly conserved across species, suggesting that the lack of Src binding may play a physiologically important and isoform-specific role.

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