Identification of agrinSN isoform and muscle-specific receptor tyrosine kinase (MuSK) [corrected] in sperm

We previously demonstrated several nicotinic acetylcholine receptor (nAChR) subunits and associated proteins in human sperm. Here, we identified in sperm for the first time two additional nAChR-associated molecules: (1) agrin(SN)Z(+) in human sperm localized in the posterior post-acrosomal, neck, and flagellar mid-piece regions; (2) a low-molecular weight isoform of muscle-specific receptor tyrosine kinase in human and mouse sperm localized in the flagellar mid-piece of human sperm.     

TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia

Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein-tyrosine kinases (PTKs) and their substrates required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls). LPS disrupted barrier integrity in a dose- and time-dependent manner, and prior broad spectrum PTK inhibition was protective. LPS increased tyrosine phosphorylation of zonula adherens proteins, VE-cadherin, gamma-catenin, and p120(ctn). Two SRC family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE-cadherin and p120(ctn) and paracellular permeability. In HMVEC-Ls, c-SRC, YES, FYN, and LYN were expressed at both mRNA and protein levels. Selective small interfering RNA-induced knockdown of c-SRC, FYN, or YES diminished LPS-induced SRC Tyr(416) phosphorylation, tyrosine phosphorylation of VE-cadherin and p120(ctn), and barrier disruption, whereas knockdown of LYN did not. For VE-cadherin phosphorylation, knockdown of either c-SRC or FYN provided total protection, whereas YES knockdown was only partially protective. For p120(ctn) phosphorylation, knockdown of FYN, c-SRC, or YES each provided comparable but partial protection. Toll-like receptor 4 (TLR4) was expressed both on the surface and intracellular compartment of HMVEC-Ls. Prior knockdown of TLR4 blocked both LPS-induced SFK activation and barrier disruption. These data indicate that LPS recognition by TLR4 activates the SFKs, c-SRC, FYN, and YES, which, in turn, contribute to tyrosine phosphorylation of zonula adherens proteins to open the endothelial paracellular pathway.     

Phosphorylation and cytoskeletal anchoring of the acetylcholine receptor by Src class protein-tyrosine kinases. Activation by rapsyn.

Src class protein-tyrosine kinases bind to and phosphorylate the nicotinic acetylcholine receptor of skeletal muscle. This study provided evidence for the functional importance of Src kinases in regulating the nicotinic acetylcholine receptor at the neuromuscular junction. Three Src class kinases, Fyn, Fyk, and Src, each formed a complex with the endplate-specific cytoskeletal protein rapsyn. In addition, cellular phosphorylation by each kinase was stimulated by rapsyn in heterologous transfected cells. Several lines of evidence supported rapsyn as a substrate for Src kinases. Most importantly, rapsyn regulation of Fyn, Fyk, and Src resulted in phosphorylation of the nicotinic acetylcholine receptor beta and delta subunits and anchoring of the receptor to the cytoskeleton. Both nicotinic acetylcholine receptor phosphorylation and cytoskeletal anchoring were blocked by the Src kinase-selective inhibitor herbimycin A. Rapsyn alone also induced a modest increase in nicotinic acetylcholine receptor phosphorylation and cytoskeletal translocation. However, inhibition by herbimycin A and a catalytically inactive dominant negative Src demonstrated that the effects of rapsyn were mediated by endogenous Src kinases. These data support the importance of Src class kinases for stabilization of the nicotinic acetylcholine receptor at the endplate during synaptic differentiation at the neuromuscular junction.     

Studies of sperm from mutant mice suggesting that two neurotransmitter receptors are important to the zona pellucida-initiated acrosome reaction

Two sperm neurotransmitter receptor/channels, the glycine receptor (GlyR) and a nicotinic acetylcholine receptor containing an alpha7 subunit (alpha7nAChR) were previously shown to be important to the mouse acrosome reaction (AR) initiated by solubilized egg zona pellucida (ZP). Here, we investigated whether sperm from homozygous mutant mice with a single amino acid mutation in the alpha subunit of their GlyR and sperm from homozygous mutant mice with an engineered disruption of the gene for the nicotinic acetylcholine receptor alpha7 subunit could undergo the AR on ZP-intact eggs. Wild-type and mutant sperm were treated with 3-quinuclidinyl benzilate (QNB), known to be an inhibitor of the ZP-initiated AR (but shown in the present work not to inhibit the acetylcholine-initiated AR). The ZP-initiated AR on ZP-intact eggs should occur only in sperm not treated with QNB. The absence of such an increase in the untreated mutant sperm would demonstrate that such sperm were unable to respond to the intact ZP. The results demonstrated for the first time that GlyR mutant sperm do not undergo the AR on ZP-intact mouse eggs, and that their ability to fertilize is inhibited by 63% in vitro. Moreover, we found that GlyR mutant sperm exhibited normal capacitation and confirmed that they not undergo the AR initiated by solubilized mouse ZP. Our studies demonstrated for the first time that sperm from mutant alpha7nAChR mice exhibit normal capacitation, do not undergo the AR in response to acetylcholine, solubilized ZP or on ZP-intact eggs, and display a 25% reduction in fertilization in vitro. This is the first genetic evidence for the importance of the alpha7nAChR in the ZP-initiated AR. While defects in either the GlyR or the alpha7nAChR completely inhibit the ZP-initiated AR, fertilization by these mutant sperm can still occur in vitro, probably due to sperm that complete spontaneous AR on the ZP.     

Primary structure and expression of beta 2: a novel subunit of neuronal nicotinic acetylcholine receptors

A new subunit, beta 2, of the neuronal nicotinic receptor family has been identified. This subunit has the structural features of a non-agonist-binding subunit. We provide evidence that beta 2 can substitute for the muscle beta 1 subunit to form a functional nicotinic receptor in Xenopus oocytes. Expression studies performed in oocytes have demonstrated that three different neuronal nicotinic acetylcholine receptors can be formed by the pairwise injection of beta 2 mRNA and each of the neuronal alpha subunit mRNAs. The beta 2 gene is expressed in PC12 cells and in areas of the central nervous system where the alpha 2, alpha 3, and alpha 4 genes are expressed. These results lead us to propose that the nervous system expresses diverse forms of neuronal nicotinic acetylcholine receptors by combining beta 2 subunits with different agonist-binding alpha subunits.     

beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis

beta-Arrestins can act as adapter molecules, coupling G-protein-coupled receptors to proteins involved in mitogenic as well as endocytic pathways. We have previously identified c-SRC as a molecule that is rapidly recruited to the beta2-adrenergic receptor in a beta-arrestin1-dependent manner. Recruitment of c-SRC to the receptor appears to be involved in pathways leading to receptor internalization and mitogen-activated protein kinase activation. This recruitment of c-SRC to the receptor involves an interaction between the amino-terminal proline-rich region of beta-arrestin1 and the Src homology 3 (SH3) domain of c-SRC, but deletion of the proline-rich domain does not totally ablate the interaction. We have found that a major interaction also exists between beta-arrestin1 and the catalytic or kinase domain (SH1) of c-SRC. We therefore hypothesized that a catalytically inactive mutant of the isolated catalytic subunit, SH1(kinase dead) (SH1(KD)), would specifically block those cellular actions of c-SRC that are mediated by beta-arrestin1 recruitment to the G-protein-coupled receptor. In contrast, the majority of cellular phosphorylations catalyzed by c-SRC, which do not involve interaction with the SH1 domain, would be predicted to be unaffected. The SH1(KD) mutant did indeed block beta2-adrenergic receptor internalization and receptor-stimulated tyrosine phosphorylation of dynamin, actions previously shown to be c-SRC-dependent. In contrast, SAM-68 and whole cell tyrosine phosphorylation by c-SRC was unaffected, indicating that the SH1(KD) mutant did not inhibit c-SRC tyrosine kinase activity in general. These results not only clarify the nature of the beta-arrestin1/c-SRC interaction but also implicate beta-arrestin1 as an important mediator of receptor internalization by recruiting tyrosine kinase activity to the cell surface to phosphorylate key endocytic intermediates, such as dynamin.     

MAP kinase stimulation by cAMP does not require RAP1 but SRC family kinases

The small G protein RAP1 and the kinase B-RAF have been proposed to link elevations of cAMP to activation of ERK/mitogen-activated protein (MAP) kinase. In order to delineate signaling pathways that link receptor-generated cAMP to the activation of MAP kinase, the human A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, was heterologously expressed in Chinese hamster ovary cells (referred as CHO-A(2A) cells). In CHO-A(2A) cells, the stimulation of the A(2A)-receptor resulted in an activation of RAP1 and formation of RAP1-B-RAF complexes. However, overexpression of a RAP1 GTPase-activating protein (RAP1GAP), which efficiently clamped cellular RAP1 in the inactive GDP-bound form, did not affect A(2A)-agonist-mediated MAP kinase stimulation. In contrast, the inhibitor of protein kinase A H89 efficiently suppressed A(2A)-agonist-mediated MAP kinase stimulation. Neither dynamin-dependent receptor internalization nor receptor-promoted shedding of matrix-bound growth factors accounted for A(2A)-receptor-dependent MAP kinase activation. PP1, an inhibitor of SRC family kinases, blunted both the A(2A)-receptor- and the forskolin-induced MAP kinase stimulation (IC(50) = 50 nm); this was also seen in PC12 cells, which express the A(2A)-receptor endogenously, and in NIH3T3 fibroblasts, in which cAMP causes MAP kinase stimulation. In the corresponding murine fibroblast cell line SYF, which lacks the ubiquitously expressed SRC family kinases SRC, YES, and FYN, forskolin barely stimulated MAP kinase; this reduction was reversed in cells in which c-SRC had been reintroduced. These findings show that activation of MAP kinase by cAMP requires a SRC family kinase that lies downstream of protein kinase A. A role for RAP1, as documented for the beta(2)-adrenergic receptor, is apparently contingent on receptor endocytosis.     

The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: beta 4

A new nicotinic acetylcholine receptor (nAChR) subunit, beta 4, was identified by screening a rat genomic library. In situ hybridization histochemistry revealed expression of the beta 4 gene in the medial habenula of adult rat brains. The primary structure of this subunit was deduced from a cDNA clone isolated from a PC12 cDNA library. Functional nAChRs were detected in Xenopus oocytes injected in pairwise combinations with in vitro synthesized RNAs encoding beta 4 and either the alpha 2, alpha 3, or alpha 4 subunit. Unlike the alpha 3 beta 2 receptor, the alpha 3 beta 4 receptor is not blocked by bungarotoxin 3.1, indicating that the beta subunit can affect the sensitivity of neuronal nAChRs to this toxin. These results extend the functional diversity of nicotinic receptors in the nervous system.     

Characterization of phosphotyrosine containing proteins at the cholinergic synapse

Tyrosine phosphorylation has been associated with several aspects of the regulation of cholinergic synaptic function, including nicotinic acetylcholine receptor (AChR) desensitization as well as the synthesis and clustering of synaptic components. While some progress has been made in elucidating the molecular events initiating such signals, the downstream targets of these tyrosine kinase pathways have yet to be characterized. In this paper we have used molecular cloning techniques to identify proteins which are tyrosine phosphorylated at the cholinergic synapse. Phosphotyrosine containing proteins (PYCPs) were isolated from the electric organ of Torpedo californica by anti-phosphotyrosine immunoaffinity chromatography. Peptide sequencing and expression cloning then identified the isolated proteins. The proteins identified included heat shock protein 90, type III intermediate filament from Torpedo electric organ, alpha-fodrin, beta-tubulin, actin and rapsyn. These tyrosine phosphorylated proteins may play a role in the regulation of synaptic function by tyrosine kinases.     

Evidence suggesting that the mouse sperm acrosome reaction initiated by the zona pellucida involves an alpha7 nicotinic acetylcholine receptor

The mammalian sperm acrosome reaction (AR) is essential to fertilization and is believed to be initiated in vivo by ZP3, a glycoprotein component of the egg zona pellucida (ZP). Recently, we reported the results of antagonist studies suggesting that a nicotinic acetylcholine receptor (nAChR) containing an alpha7 subunit (alpha7nAChR) plays a role in the human sperm AR initiated by recombinant human ZP3 or by acetylcholine (ACh). Here, we show that ACh can initiate the mouse sperm AR and that antagonists of the nAChR inhibit the AR initiated by ACh or by ZP obtained from ovarian oocytes (isolated heat-solubilized mouse ZP). Preincubation with three antagonists of the nAChR, alpha-bungarotoxin (100 nM), alpha-conotoxin IMI (100 nM), and methyllycaconitine (100 nM), significantly blocked AR initiation by ACh or by isolated heat-solubilized mouse ZP (P 相似文献   

Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking alpha (alpha2-alpha4, alpha6) and beta (beta2, beta4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (beta4_alpha2, beta4_alpha3, beta4_alpha4, and beta2_alpha4) when coexpressed with the corresponding beta monomer subunit. All tandems expressed when injected alone, except for beta4_alpha3, which produced functional channels only together with beta4 monomer and was chosen for further characterization. These channels produced from beta4_alpha3 tandem constructs plus beta4 monomer were identical with receptors expressed from monomer alpha3 and beta4 constructs in acetylcholine sensitivity and in the number of alpha and beta subunits incorporated in the channel gate. However, separately mutating the beta subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of beta subunits from the tandem and one from the beta monomer construct, whereas the rest incorporated two or three beta monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.     

The chaperone protein 14-3-3eta interacts with the nicotinic acetylcholine receptor alpha 4 subunit. Evidence for a dynamic role in subunit stabilization

By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.     

Sperm epidermal growth factor receptor (EGFR) mediates α7 acetylcholine receptor (AChR) activation to promote fertilization

To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca(2+) influx, and the acrosome reaction.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.     

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.     

Regulation of c-SRC activity and function by the adapter protein CAS

SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130(CAS) (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.