Evidence for cardiac sodium-calcium exchanger association with caveolin-3

The interaction of cardiac Na+-Ca2+ exchange (NCX1) with caveolin proteins was investigated in sarcolemmal vesicles. Western blots of sarcolemmal vesicles revealed the presence of caveolin-1, -2, and -3. NCX1 co-fractionated more closely with caveolin-3 than caveolin-1 on sucrose density gradients. NCX1 has five possible caveolin-binding motifs and NCX1 co-precipitated specifically with caveolin-3. Molecular sieve column chromatography indicated that this co-precipitation was not due to incomplete solubilization of lipid raft microdomains. Cholesterol chelation in vesicles decreased NCX1 transport activity and caveolin-3 co-precipitation. NCX1 may play a role in caveolar transmembrane signaling in addition to its role in excitation-contraction coupling.     

Striatin, a calmodulin-dependent scaffolding protein, directly binds caveolin-1.

Caveolins are scaffolding proteins able to collect on caveolae a large number of signalling proteins bearing a caveolin-binding motif. The proteins of the striatin family, striatin, SG2NA, and zinedin, are composed of several conserved, collinearly aligned, protein-protein association domains, among which a putative caveolin-binding domain [Castets et al. (2000) J. Biol. Chem. 275, 19970-19977]. They are associated in part with membranes. These proteins are mainly expressed within neurons and thought to act both as scaffolds and as Ca(2+)-dependent signalling proteins [Bartoli et al. (1999) J. Neurobiol. 40, 234-243]. Here, we show that (1) rat brain striatin, SG2NA and zinedin co-immunoprecipitate with caveolin-1; (2) all are pulled down by glutathione-S-transferase (GST)-caveolin-1; (3) a fragment of recombinant striatin containing the putative caveolin-binding domain binds GST-caveolin-1. Hence, it is likely that the proteins of the striatin family are addressed to membrane microdomains by their binding to caveolin, in accordance with their putative role in membrane trafficking [Baillat et al. (2001) Mol. Biol. Cell 12, 663-673].     

A novel insulin receptor-signaling platform and its link to insulin resistance and type 2 diabetes

Insulin-induced insulin receptor (IR) tyrosine kinase activation and insulin cell survival responses have been reported to be under the regulation of a membrane associated mammalian neuraminidase-1 (Neu1). The molecular mechanism(s) behind this process is unknown. Here, we uncover a novel Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B G-protein coupled receptor (GPCR), which is essential for insulin-induced IR activation and cellular signaling. Neu1, MMP-9 and neuromedin B GPCR form a complex with IRβ subunit on the cell surface. Oseltamivir phosphate (Tamiflu®), anti-Neu1 antibodies, broad range MMP inhibitors piperazine and galardin (GM6001), MMP-9 specific inhibitor (MMP-9i), and GPCR neuromedin B specific antagonist BIM-23127 dose-dependently inhibited Neu1 activity associated with insulin stimulated rat hepatoma cells (HTCs) that overly express human IRs (HTC-IR). Tamiflu, anti-Neu1 antibodies and MMP-9i attenuated phosphorylation of IRβ and insulin receptor substrate-1 (IRS1) associated with insulin-stimulated cells. Olanzapine, an antipsychotic agent associated with insulin resistance, induced Neu3 sialidase activity in WG544 or 1140F01 human sialidosis fibroblast cells genetically defective in Neu1. Neu3 antagonist 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and anti-Neu3 antibodies inhibited sialidase activity associated with olanzapine treated murine Neu4 knockout macrophage cells. Olanzapine attenuated phosphorylation of IGF-R and IRS1 associated with insulin-stimulated human wild-type fibroblast cells. Our findings identify a novel insulin receptor-signaling platform that is critically essential for insulin-induced IRβ tyrosine kinase activation and cellular signaling. Olanzapine-induced Neu3 sialidase activity attenuated insulin-induced IGF-R and IRS1 phosphorylation contributing to insulin resistance.     

P-glycoprotein in blood-brain barrier endothelial cells: interaction and oligomerization with caveolins

P-glycoprotein (P-gp), an adenosine triphosphate (ATP)-binding cassette transporter which acts as a drug efflux pump, is highly expressed at the blood-brain barrier (BBB) where it plays an important role in brain protection. Recently, P-gp has been reported to be located in the caveolae of multidrug-resistant cells. In this study, we investigated the localization and the activity of P-gp in the caveolae of endothelial cells of the BBB. We used an in vitro model of the BBB which is formed by co-culture of bovine brain capillary endothelial cells (BBCEC) with astrocytes. Caveolar microdomains isolated from BBCEC are enriched in P-gp, cholesterol, caveolin-1, and caveolin-2. Moreover, P-gp interacts with caveolin-1 and caveolin-2; together, they form a high molecular mass complex. P-gp in isolated caveolae is able to bind its substrates, and the caveolae-disrupting agents filipin III and nystatin decrease P-gp transport activity. In addition, mutations in the caveolin-binding motif present in P-gp reduced the interaction of P-gp with caveolin-1 and increased the transport activity of P-gp. Thus, P-gp expressed at the BBB is mainly localized in caveolae and its activity may be modulated by interaction with caveolin-1.     

Molecular characterization of membrane type and ganglioside-specific sialidase (Neu3) expressed in E. coli

Endogenous expression of human membrane type ganglioside sialidase (Neu3) was examined in various cell lines including NB-1, U87MG, SK-MEL-2, SK-N-MC, HepG2, Hep3B, Jurkat, HL-60, K562, ECV304, Hela and MCF-7. Expression was detected in the neuroblastoma cell lines NB-1 and SK-N-MC, and also in erythroleukemia K562 cells, but not in any other cells. We isolated a Neu3 cDNA from K562 cells and expressed a His-tagged derivative in a bacterial expression system. The purified recombinant product of approximately 48 kDa had sialidase activity toward 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH of the purified Neu3 protein for GD3 ganglioside was 4.5. The enzyme also efficiently hydrolyzed GD3, GD1a, GD1b and GM3 whereas sialyllactose, 4MU-NeuAc, GM1 and GM2 were poor substrates, and it had no activity against sialylated glycoproteins such as fetuin, transferrin and orosomucoid. We conclude that the sialidase activity of Neu3 is specific for gangliosides.     

Differential expression of endogenous sialidases of human monocytes during cellular differentiation into macrophages

Sialidases are enzymes that influence cellular activity by removing terminal sialic acid from glycolipids and glycoproteins. Four genetically distinct sialidases have been identified in mammalian cells. In this study, we demonstrate that three of these sialidases, lysosomal Neu1 and Neu4 and plasma membrane-associated Neu3, are expressed in human monocytes. When measured using the artificial substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (4-MU-NANA), sialidase activity of monocytes increased up to 14-fold per milligram of total protein after cells had differentiated into macrophages. In these same cells, the specific activity of other cellular proteins (e.g. beta-galactosidase, cathepsin A and alkaline phosphatase) increased only two- to fourfold during differentiation of monocytes. Sialidase activity measured with 4-MU-NANA resulted from increased expression of Neu1, as removal of Neu1 from the cell lysate by immunoprecipitation eliminated more than 99% of detectable sialidase activity. When exogenous mixed bovine gangliosides were used as substrates, there was a twofold increase in sialidase activity per milligram of total protein in monocyte-derived macrophages in comparison to monocytes. The increased activity measured with mixed gangliosides was not affected by removal of Neu1, suggesting that the expression of a sialidase other than Neu1 was present in macrophages. The amount of Neu1 and Neu3 RNAs detected by real time RT-PCR increased as monocytes differentiated into macrophages, whereas the amount of Neu4 RNA decreased. No RNA encoding the cytosolic sialidase (Neu2) was detected in monocytes or macrophages. Western blot analysis using specific antibodies showed that the amount of Neu1 and Neu3 proteins increased during monocyte differentiation. Thus, the differentiation of monocytes into macrophages is associated with regulation of the expression of at least three distinct cellular sialidases, with specific up-regulation of the enzyme activity of only Neu1.     

The plasma membrane ganglioside sialidase cofractionates with markers of lipid rafts

Gangliosides of the plasma membrane are important modulators of cellular functions. Recent reports have shown their enrichment in glycosphingolipid-containing membrane microdomains, called glycosphingolipid-signaling domain or rafts, which can be isolated due to their insolubility in Triton X-100 and flotation through a sucrose gradient. In previous work on neuroblastoma cells we had found that a ganglioside-specific sialidase activity of the plasma membrane controlled proliferation and differentiation through selective ganglioside desialylation. Assuming the ganglioside sialidase to be close to its substrates in the membrane, we investigated its association with detergent-insoluble microdomains in the neuroblastoma cell line SK-N-MC. The results show that the ganglioside sialidase codistributes with the raft markers ganglioside GM1, flotillin, src family kinases, and glycosylphosphatidylinositol-anchored proteins in a fraction containing about 2% of cellular protein. The association of the ganglioside sialidase with glycosphingolipid-enriched membrane fractions therefore is in support of a role of this glycosidase in ganglioside-dependent signaling processes.     

Computational 3-D modeling and site-directed mutation of an antibody that binds Neu2en5Ac, a transition state analogue of a sialic acid.

An antibody against a transition state analog (TSA) may share some common features with an enzyme that produces such a transition state. SIC172 antibody binds specifically to Neu2en5Ac, a TSA of Neu5Ac in the sialidase reaction, but has no catalytic activity. To understand how the antibody recognizes Neu2en5Ac and to find out if it is possible to convert it to a catalytic antibody, we made and sequenced the SIC172 ScFv, and constructed a 3-D model of it. The VH-CDR3 contains a unique sequence with Cys at H95. The 3-D model showed that Cys-H95 is exposed inside the antigen-binding cavity. After affinity docking, 4 types emerged. In type I, the carboxyl group of Neu2en5Ac is located near the Cys-H95 and neighboring positively charged residues. The change of Cys-H95 to Asp by site-directed mutation decreased the binding activity, while a change to Arg did not. These and other mutation experiments, and further modeling of mutant Fv, support the 3-D model and docking type I. A comparison with sialidase indicates that SIC172 antibody appears to have some groups of residues that are conserved at the active site of the enzyme. The possibility of Neu2en5Ac-binding antibody being converted to a catalytic antibody is discussed.     

Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase

The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC₅₀ of 1.2?μM compared to an IC₅₀ of 1015?μM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFκB and the production of nitric oxide and pro-inflammatory IL-6 and TNFα cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.     

Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for neurotrophin activation of Trk receptors and cellular signaling

Neurotrophin-induced Trk tyrosine kinase receptor activation and neuronal cell survival responses have been reported to be under the control of a membrane associated sialidase. Here, we identify an unprecedented membrane sialidase mechanism initiated by nerve growth factor (NGF) binding to TrkA to potentiate GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live primary neurons and TrkA- and TrkB-expressing cell lines. Central to this process is that Neu1/MMP-9 complex is bound to TrkA on the cell surface of naïve primary neurons and TrkA-expressing cells. Tamiflu completely blocks this sialidase activity in live TrkA-PC12 cells treated with NGF with an IC₅₀ of 3.876 μM with subsequent inhibition of Trk activation in primary neurons and neurite outgrowth in TrkA-PC12 cells. Our findings uncover a Neu1 and MMP-9 cross-talk on the cell surface that is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and cellular signaling.     

Low molecular weight protein tyrosine phosphatase and caveolin-1: interaction and isoenzyme-dependent regulation

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are small enzymes that are ubiquitous in many organisms. They are important in biological processes such as cell proliferation, adhesion, migration, and invasiveness. LMW-PTP is expressed in mammalian cells as two isoforms (IF1 and IF2) originating through alternative splicing. We have previously shown that IF2 targets lipid rafts called caveolae and interacts with caveolin-1, their major structural protein. Caveolae are cholesterol- and sphingolipid-rich membrane microdomains that have been implicated in a variety of cellular functions, including signal transduction events. Caveolin-1 contains a scaffolding region that contributes to the binding of the protein to the plasma membrane and mediates protein omo- and etero-oligomerization. Interaction of many signaling molecules with the scaffolding domain sequesters them into caveolae and inhibits or suppresses their activities. Caveolin-interacting proteins usually have a typical sequence motif, also present in all the LMW-PTPs, which is characterized by aromatic or large hydrophobic residues in specific positions. We have examined here the interaction of the LMW-PTP isoforms with caveolin-1 and its molecular mechanism, together with the consequences for their tyrosine phosphatase activities. We found that IF1 and IF2 are both capable of interacting with defined regions of caveolin-1 and that their putative caveolin binding sequence motif is not responsible for the association. The formation of LMW-PTP/caveolin-1 complexes is accompanied by modulation of the enzyme activities, and the inhibitory effect elicited against IF1 is stronger than that against IF2. The caveolin scaffolding domain is directly involved in the observed phenomena.     

The sonic hedgehog receptor patched associates with caveolin-1 in cholesterol-rich microdomains of the plasma membrane

The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, we hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein.     

Caveolin-1 is transiently dephosphorylated by shear stress-activated protein tyrosine phosphatase mu

Endothelial cells are subjected to hemodynamic shear stress, which regulates multiple vascular functions partially by the caveolin-1-dependent mechanisms. Caveolin-1 is a principal protein in the plasma membrane microdomains called caveolae and interacts with various signaling molecules. Recently, caveolin-1 was elucidated to be phosphorylated on tyrosine 14. However, it is not known how phosphorylation of caveolin-1 is controlled in endothelium. In this study, we found that caveolin-1 is phosphorylated by p38 mitogen-activated protein kinase (MAPK) under a static condition. When endothelial cells were exposed to shear stress, caveolin-1 was transiently dephosphorylated. Since the activity of p38 MAPK was not affected by shear stress, the shear-dependent dephosphorylation of caveolin-1 was not mediated by p38 MAPK. Of interest, sodium orthovanadate, an inhibitor for phosphatases, blocked the shear-dependent dephosphorylation of caveolin-1. We also observed that protein tyrosine phosphatase mu was transiently activated by shear stress, suggesting its role in the dephosphorylation of caveolin-1.     

Some protein tyrosine phosphatases target in part to lipid rafts and interact with caveolin-1

A profile-based search of the SWISS-PROT database reveals that most protein tyrosine phosphatases (PTPs) contain at least one caveolin-1-binding motif. To ascertain if the presence of caveolin-binding motif(s) in PTPs corresponds to their actual localization in caveolin-1-enriched membrane fractions, we performed subcellular fractionating experiments. We found that all tested PTPs (PTP1B, PTP1C, SHPTP2, PTEN, and LAR) are actually localized in caveolin-enriched membrane fractions, despite their distribution in other subcellular sites, too. More than 1/2 of LAR and about 1/4 of SHPTP2 and PTP-1C are localized in caveolin-enriched membrane fractions whereas, in these fractions, PTP-1B and PTEN are poorly concentrated. Co-immunoprecipitation experiments with antibodies specific for each tested PTP demonstrated that all five phosphatases form molecular complexes with caveolin-1 in vivo. Collectively, our findings propose that particular PTPs could perform some of their cellular actions or are regulated by recruitment into caveolin-enriched membrane fractions.     

Possible association of Neu2 with plasma membrane fraction from mouse thymus exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5

Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4MU‐Neu5Ac as a substrate. The main purpose of the present study was to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP‐40 or DOC/NP‐40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same pI as that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10 mM Tris‐buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane‐rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2‐transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.     

Insulin-like growth factor 1 signaling regulates cytosolic sialidase Neu2 expression during myoblast differentiation and hypertrophy

Cytosolic sialidase (neuraminidase 2; Neu2) is an enzyme whose expression increases during myoblast differentiation. Here we show that insulin-like growth factor 1 (IGF1)-induced hypertrophy of myoblasts notably increases Neu2 synthesis by activation of the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (P13K/AKT/mTOR) pathway, whereas the proliferative effect mediated by activation of the extracellular regulated kinase 1/2 (ERK1/2) pathway negatively contributed to Neu2 activity. Accordingly, the differentiation L6MLC/IGF-1 cell line, in which the forced postmitotic expression of insulin-like growth factor 1 stimulates a dramatic hypertrophy, was accompanied by a stronger Neu2 increase. Indeed, the hypertrophy induced by transfection of a constitutively activated form of AKT was able to induce high Neu2 activity in C2C12 cells, whereas the transfection of a kinase-inactive form of AKT prevented myotube formation, triggering Neu2 downregulation. Neu2 expression was strictly correlated with IGF-1 signaling also in C2 myoblasts overexpressing the insulin-like growth factor 1 binding protein 5 and therefore not responding to endogenously produced insulin-like growth factor 1. Although Neu2-transfected myoblasts exhibited stronger differentiation, we demonstrated that Neu2 overexpression does not override the block of differentiation mediated by PI3 kinase and mTOR inhibitors. Finally, Neu2 overexpression did not modify the ganglioside pattern of C2C12 cells, suggesting that glycoproteins might be the target of Neu2 activity. Taken together, our data demonstrate that IGF-1-induced differentiation and hypertrophy are driven, at least in part, by Neu2 upregulation and further support the significant role of cytosolic sialidase in myoblasts.     

Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase

A direct link between receptor glycosylation and activation following natural ligand interaction has not been observed. Here, we discover a membrane sialidase-controlling mechanism that depends on ligand binding to its receptor to induce enzyme activity which targets and desialylates the receptor and, consequently, causes the induction of receptor dimerization and activation. We also identify a specific sialyl alpha-2,3-linked beta-galactosyl sugar residue of TrkA tyrosine kinase receptor, which is rapidly targeted and hydrolyzed by the sialidase. Trk-expressing cells and primary cortical neurons following stimulation with specific neurotrophic growth factors express a vigorous membrane sialidase activity. Neuraminidase inhibitors, Tamiflu, BCX1812, and BCX1827, block sialidase activity induced by nerve growth factor (NGF) in TrkA-PC12 cells and by brain-derived neurotrophic factor (BDNF) in primary cortical neurons. In contrast, the neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, specific for plasma membrane ganglioside Neu3 and Neu2 sialidases has no inhibitory effect on NGF-induced pTrkA. The GM1 ganglioside specific cholera toxin subunit B applied to TrkA-PC12 cells has no inhibitory effect on NGF-induced sialidase activity. Neurite outgrowths induced by NGF-treated TrkA-PC12 and BDNF-treated PC12(nnr5) stably transfected with TrkB receptors (TrkB-nnr5) cells are significantly inhibited by Tamiflu. Our results establish a novel mode of regulation of receptor activation by its natural ligand and define a new function for cellular sialidases.     

Biased G protein-coupled receptor agonism mediates Neu1 sialidase and matrix metalloproteinase-9 crosstalk to induce transactivation of insulin receptor signaling

G protein-coupled receptors (GPCR) can participate in a number of signaling pathways, and this property led to the concept of biased GPCR agonism. Agonists, antagonists and allosteric modulators can bind to GPCRs in different ways, creating unique conformations that differentially modulate signaling through one or more G proteins. A unique neuromedin B (NMBR) GPCR-signaling platform controlling mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP9) crosstalk has been reported in the activation of the insulin receptor (IR) through the modification of the IR glycosylation. Here, we propose that there exists a biased GPCR agonism as small diffusible molecules in the activation of Neu1-mediated insulin receptor signaling. GPCR agonists bombesin, bradykinin, angiotensin I and angiotensin II significantly and dose-dependently induce Neu1 sialidase activity and IR activation in human IR-expressing rat hepatoma cell lines (HTC-IR), in the absence of insulin. Furthermore, the GPCR agonist-induced Neu1 sialidase activity could be specifically blocked by the NMBR inhibitor, BIM-23127. Protein expression analyses showed that these GPCR agonists significantly induced phosphorylation of IRβ and insulin receptor substrate-1 (IRS1). Among these, angiotensin II was the most potent GPCR agonist capable of promoting IRβ phosphorylation in HTC-IR cells. Interestingly, treatment with BIM-23127 and Neu1 inhibitor oseltamivir phosphate were able to block GPCR agonist-induced IR activation in HTC cells in vitro. Additionally, we found that angiotensin II receptor (type I) exists in a multimeric receptor complex with Neu1, IRβ and NMBR in naïve (unstimulated) and stimulated HTC-IR cells with insulin, bradykinin, angiotensin I and angiotensin II. This complex suggests a molecular link regulating the interaction and signaling mechanism between these molecules on the cell surface. These findings uncover a biased GPCR agonist-induced IR transactivation signaling axis, mediated by Neu1 sialidase and the modification of insulin receptor glycosylation.