Regulation of nicotinic acetylcholine receptors by tyrosine kinases in the peripheral and central nervous system: same players, different roles.

Nicotinic acetylcholine receptors (nAChRs) exist in many subtypes and are found in the peripheral and central nervous system where they mediate or modulate synaptic transmission. We review how tyrosine phosphorylation and kinases regulate muscle and neuronal nAChRs. Interestingly, although some of the same kinase players interact with the various receptor subtypes, the functional consequences are different. While concerted action of MuSK, Abl- and Src-family kinases (SFKs) regulates the synaptic distribution of nAChRs at the neuromuscular junction, SFKs activate heteromeric neuronal nAChRs in adrenal chromaffin cells, thereby enhancing catecholamine secretion. In contrast, the activity of homomeric neuronal nAChRs, as found in the hippocampus, is negatively regulated by tyrosine phosphorylation and SFKs. It appears that tyrosine kinases provide the means to regulate all nAChRs; but the functional consequences, even those caused by the same kinase family, are specific for each receptor subtype and location.

Sindrome de ovario poliquistico: presentacion clinica, bioquimica y ultrasonografica

En forma retrospectiva se efectúa una revisión de 115 historias clínicas de pacientes con Síndrome de Ovario Poliquístico (SOP) que consultan en la Unidad de Endocrinología del Servicio de Ginecología del Hospital San José en el periodo comprendido entre los años 1996 y 2002. Se determinó la prevalencia y se estableció una caracterización demográfica, hormonal y ultrasonográfica de estas pacientes. Destaca la presencia de un alto porcentaje de obesidad que alcanzó el 63% y una insulinoresistencia del orden del 76%. La LH se encontró elevada en el 47% de nuestras pacientes y la testosterona total y libre mostraron un bajo porcentaje de incremento (11% y 27% respectivamente). La Ultrasonografía mostró patrones característicos de SOP, de acuerdo a los criterios estandarizados actuales, en alrededor del 70% de los casos. Estos hallazgos nos inducen a priorizar el estudio de estas pacientes en base a LH y a insulinoresistencia por sobre los niveles de andrógenos. Desde el punto de vista metabólico se encontró un bajo porcentaje de Hipertensión Arterial y Diabetes Mellitus II (2,6 y 6,1% respectivamente).

Neuromuscular synaptogenesis: clustering of acetylcholine receptors revisited.

Clustering of neurotransmitter receptors in the postsynaptic membrane is critical for efficient synaptic transmission. During neuromuscular synaptogenesis, clustering of acetylcholine receptors (AChRs) is an early sign of postsynaptic differentiation. Recent studies have revealed that the earliest AChR clusters can form in the muscle independent of motorneurons. Neurally released agrin, acting through the muscle-specific kinase MuSK and rapsyn, then causes further clustering and localization of clusters underneath the nerve terminal. AChRs themselves are required for agrin-induced clustering of several postsynaptic proteins, most notably rapsyn. Once formed, AChR clusters are stabilized by several tyrosine kinases and by components of the dystrophin/utrophin glycoprotein complex, some of which also direct postnatal synaptic maturation such as formation of postjunctional folds. This review summarizes these recent results about AChR clustering, which indicate that early clustering can occur in the absence of nerves, that AChRs play an active role in the clustering process and that partly different mechanisms direct formation versus stabilization of AChR clusters.

Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins.

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, alpha-dystrobrevin and utrophin, but failed to aggregate AChRs, alpha- and beta-dystroglycan, syntrophin isoforms and rapsyn, an AChR-anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn efficiently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myofibers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.

Src, Fyn, and Yes are not required for neuromuscular synapse formation but are necessary for stabilization of agrin-induced clusters of acetylcholine receptors.

Mice deficient in src and fyn or src and yes move and breathe poorly and die perinatally, consistent with defects in neuromuscular function. Src and Fyn are associated with acetylcholine receptors (AChRs) in muscle cells, and Src and Yes can act downstream of ErbB2, suggesting roles for Src family kinases in signaling pathways regulating neuromuscular synapse formation. We studied neuromuscular synapses in src(-/-); fyn(-/-) and src(-/-); yes(-/-) mutant mice and found that muscle development, motor axon pathfinding, clustering of postsynaptic proteins, and synapse-specific transcription are normal in these double mutants, showing that these pairs of kinases are not required for early steps in synapse formation. We generated muscle cell lines lacking src and fyn and found that neural agrin and laminin-1 induced normal clustering of AChRs and that agrin induced normal tyrosine phosphorylation of the AChR beta subunit in the absence of Src and Fyn. Another Src family member, most likely Yes, was associated with AChRs and phosphorylated by agrin in myotubes lacking Src and Fyn, indicating that Yes may compensate for the loss of Src and Fyn. Nevertheless, PP1 and PP2, inhibitors of Src-class kinases, did not inhibit agrin signaling, suggesting that Src class kinase activity is dispensable for agrin-induced clustering and tyrosine phosphorylation of AChRs. AChR clusters, however, were less stable in myotubes lacking Src and Fyn but not in PP1- or PP2-treated wild-type cells. These data show that the stabilization of agrin-induced AChR clusters requires Src and Fyn and suggest that the adaptor activities, rather than the kinase activities, of these kinases are essential for this stabilization.

Agrin-induced activation of acetylcholine receptor-bound Src family kinases requires Rapsyn and correlates with acetylcholine receptor clustering.

During neuromuscular synaptogenesis, neurally released agrin induces aggregation and tyrosine phosphorylation of acetylcholine receptors (AChRs) by acting through both the receptor tyrosine kinase MuSK (muscle-specific kinase) and the AChR-associated protein, rapsyn. To elucidate this signaling mechanism, we examined tyrosine phosphorylation of AChR-associated proteins, particularly addressing whether agrin activates Src family kinases bound to the AChR. In C2 myotubes, agrin induced tyrosine phosphorylation of these kinases, of AChR-bound MuSK, and of the AChR beta and delta subunits, as observed in phosphotyrosine immunoblotting experiments. Kinase assays revealed that the activity of AChR-associated Src kinases was increased by agrin, whereas phosphorylation of the total cellular kinase pool was unaffected. In both rapsyn-deficient myotubes and staurosporine-treated C2 myotubes, where AChRs are not clustered, agrin activated MuSK but did not cause either Src family or AChR phosphorylation. In S27 mutant myotubes, which fail to aggregate AChRs, no agrin-induced phosphorylation of AChR-bound Src kinases, MuSK, or AChRs was observed. These results demonstrate first that agrin leads to phosphorylation and activation of AChR-associated Src-related kinases, which requires rapsyn, occurs downstream of MuSK, and causes AChR phosphorylation. Second, this activation intimately correlates with AChR clustering, suggesting that these kinases may play a role in agrin-induced AChR aggregation by forming an AChR-bound signaling cascade.

Roles of rapsyn and agrin in interaction of postsynaptic proteins with acetylcholine receptors.

At the neuromuscular junction, aggregates of acetylcholine receptors (AChRs) are anchored in the muscle membrane by association with rapsyn and other postsynaptic proteins. We have investigated the interactions between the AChR and these proteins in cultured C2 myotubes before and after treatment with agrin, a nerve-derived protein that induces AChRs to cluster. When AChRs were isolated from detergent extracts of untreated C2 myotubes, they were associated with rapsyn and, to a lesser degree, with utrophin, beta-dystroglycan, MuSK, and src-related kinases, but not with syntrophin. Treatment with agrin increased the association of AChRs with MuSK, a receptor tyrosine kinase that forms part of the agrin receptor complex, without affecting other interactions. Analysis of rapsyn-deficient myotubes, which do not form protein clusters in response to agrin, revealed that rapsyn is required for association of the AChR with utrophin and beta-dystroglycan, and for the agrin-induced increase in association with MuSK, but not for constitutive interactions with MuSK and src-related kinases. In rapsyn -/- myotubes, agrin caused normal tyrosine phosphorylation of AChR-associated and total MuSK, whereas phosphorylation of the AChR beta subunit, both constitutive and agrin-induced, was strongly reduced. These results show first that aneural myotubes contain preassembled AChR protein complexes that may function in the assembly of the postsynaptic apparatus, and second that rapsyn, in addition to its role in AChR phosphorylation, mediates selected protein interactions with the AChR and serves as a link between the AChR and the dystrophin/utrophin glycoprotein complex.

Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle.

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.

Functional interaction of Src family kinases with the acetylcholine receptor in C2 myotubes.

Tyrosine phosphorylation of the beta subunit of the acetylcholine receptor (AChR) has been postulated to play a role in AChR clustering during development of the neuromuscular junction. We have investigated the mechanism of this phosphorylation in mammalian C2 myotubes and report that the tyrosine kinase Src binds and phosphorylates glutathione S-transferase fusion proteins containing the N-terminal half of the cytoplasmic loop of the beta subunit. No binding occurs to the related kinases Fyn or Yes or to the corresponding regions from the gamma and delta subunits. Furthermore, AChRs affinity-isolated from C2 myotubes using alpha-bungarotoxin-Sepharose were specifically associated with Src and Fyn and had tyrosine-phosphorylated beta subunits. We suggest that AChRs are initially phosphorylated by Src and subsequently bind Fyn in a phosphotyrosine-dependent manner. These interactions are likely to play an important role in construction of the specialized postsynaptic membrane during synaptogenesis.

Dynamic interactions of the asialoglycoprotein receptor subunits with coated pits. Enhanced interactions of H2 following association with H1.

Lateral mobility studies comparing native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, can measure membrane protein-coated pit interactions in intact cells (Fire, E., Zwart, D., Roth, M. G., and Henis, Y. I. (1991) J. Cell Biol. 115, 1585-1594). We applied this approach to study the interactions of the H1 and H2 human asialoglycoprotein receptor subunits with coated pits. The lateral mobilities of singly expressed and coexpressed H1 and H2B (the H2 species that reaches the cell surface) were measured by fluorescence photobleaching recovery. They were compared with mutant proteins, H1(5A) (Tyr-5 replaced by Ala) and H2(5A) (Phe-5 replaced by Ala). While the mobile fractions of H1, H2B, and their mutants were similar, the lateral diffusion rate (measured by D, the lateral diffusion coefficient) was significantly slower for H1, whether expressed alone or with H2B. Coexpression with H1 reduced D of H2B to that of H1. Disruption of the clathrin lattices by hypertonic medium elevated D of H1, H1(5A), H2B, and H2(5A) to the same final level, without affecting their mobile fractions. Cytosol acidification, which retains altered clathrin lattices attached to the membrane and prevents coated vesicle formation, immobilized part of the H1 molecules, reflecting stable entrapment in "frozen" coated pits. H1(5A), H2B, and H2(5A) were not affected; however, coexpression of H2B with H1 conferred the sensitivity to cytosol acidification on H2B. Our results suggest that H1 lateral mobility is inhibited by dynamic interactions with coated pits in which Tyr-5 is involved. H2B resembles H1(5A) rather than H1, and its interactions with coated pits are weaker; efficient interaction of H2B with coated pits depends on complex formation with H1.

The two subunits of the asialoglycoprotein receptor contain different sorting information.

The human asialoglycoprotein receptor, an endocytic transport receptor of the basolateral surface of hepatocytes, is a hetero-oligomer of two homologous subunits H1 and H2. The cytoplasmic domain of H1 has been shown previously to contain a tyrosine-based signal for endocytosis and basolateral sorting. Here, we have investigated sorting determinants within subunit H2 and their contribution to the targeting of the hetero-oligomeric receptor complex. Despite extensive sequence homology, H2 expressed separately in fibroblast cells was endocytosed poorly, and mutation of phenylalanine 5 (corresponding to the critical tyrosine in H1) did not further reduce internalization. Consistent with this observation, ligand uptake by receptors composed of H1 lacking tyrosine 5 and H2 was inefficient. With respect to polarized transport in Madin-Darby canine kidney cells, H2 could not be analyzed separately, because in the absence of H1 subunit H2 was completely degraded intracellularly. Coexpression of both subunits yielded ligand-binding receptors located specifically on the basolateral surface. The mutant H1(5A) (tyrosine 5 replaced by alanine) is approximately 55% apical in the absence of H2. In cells expressing H1(5A) together with H2, however, subunit H2 directed receptor complexes exclusively to the basolateral domain. Phenylalanine 5 is not essential for basolateral transport. Thus, whereas the endocytosis signal of the hetero-oligomeric asialoglycoprotein receptor resides exclusively in subunit H1, polarized transport to the basolateral domain of Madin-Darby canine kidney cells may involve two signals, only one of which is active for endocytosis.

Characterization of diazepam submicron emulsion interface: role of oleic acid.

Oleic acid markedly improved the physical stability of a diazepam submicron emulsion. The zeta potential of the emulsion increased with increased oleic acid concentration. This effect suggested that adjustment of the diazepam submicron emulsion pH to 7.8-8.0 led to the ionization of oleic acid molecules at the oil/water (o/w) interface without being excluded from the surface regions of the oil droplets. TEM freeze-fracturing examination revealed that a mixed-emulsifier monolayer film was established at the o/w interface of the submicron emulsion. No liquid crystal or liposome formation was detected. This was confirmed by the results of phosphatidylethanolamine surface labelling at the o/w interface of the emulsion. The improved stability properties conferred to the emulsion by oleic acid should be attributed not only to the zeta potential increase, but also to the strengthening of the molecular interactions occurring between phospholipid and poloxamer emulsifiers in the presence of an ionized form of oleic acid at the o/w interface of the emulsified oily droplets as evidenced in independent monolayer studies.

Related signals for endocytosis and basolateral sorting of the asialoglycoprotein receptor.

The major subunit of the human asialoglycoprotein receptor contains signals for efficient endocytosis and specific basolateral expression in polarized Madin-Darby canine kidney cells, both of which are located within its 40-residue cytoplasmic domain. The aromatic residue in this segment, tyrosine 5, which is necessary for efficient clustering into clathrin-coated pits at the plasma membrane, is also necessary for exclusive basolateral delivery. Mutation of this residue to alanine resulted in a nonpolar expression of the protein. Replacement of tyrosine 5 with phenylalanine yielded almost wild-type rates of endocytosis as well as specific basolateral expression, indicating that tyrosine phosphorylation is not essential for either sorting step. The close similarity between the two sorting signals was further corroborated by deletion mutants showing that the amino-terminal 10 residues of the cytoplasmic domain are sufficient for basolateral polarity and efficient endocytosis. The kinetics of appearance of newly synthesized wild-type and mutant receptor protein at the apical and basolateral surfaces indicate that these proteins are sorted intracellularly and are transported directly to the respective domains. Mutants affected in basolateral sorting lost polarity, i.e, appeared to similar extents on both surfaces, indicating that there is no significant apical sorting information elsewhere in the protein. The close correlation between endocytosis and basolateral polarity suggests common recognition mechanisms at the plasma membrane and in the trans-Golgi network.

Endocytosis by the asialoglycoprotein receptor is independent of cytoplasmic serine residues.

The human asialoglycoprotein (ASGP) receptor, like most other plasma membrane receptors, has previously been shown to be phosphorylated at serine residues within the cytoplasmic domain. Phorbol esters, which activate protein kinase C, cause hyperphosphorylation and down-regulation of the ASGP receptor in HepG2 cells. To test the importance of serine residues for receptor traffic and function, we have mutated all the cytoplasmic serines of the two receptor subunits H1 (at positions 16 and 37) and H2 (at positions 12, 13, and 55) to alanines or glycines. Stable transfected fibroblast cell lines expressing either mutant H1 alone or both mutant subunits together were created and compared to cell lines expressing the respective wild-type proteins. Mutant and wild-type subunits were found to have very similar distributions between the cell surface and intracellular compartments. Constitutive internalization of H1 alone and ligand uptake and degradation by cells expressing both receptor subunits were not affected by the mutations. Cytoplasmic serines and serine phosphorylation are thus not essential for receptor function and intracellular traffic. Analysis of individual serine mutations identified serine-12 of subunit H2 as the major site of phosphorylation in the ASGP receptor.

Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits.

The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization was reduced by a factor of four. The residual rate of endocytosis, however, was still significantly higher than that of resident plasma membrane proteins. Upon acidification of the cytoplasm, which specifically inhibits the formation of clathrin-coated vesicles but not uptake of the fluid phase marker Lucifer yellow, residual endocytosis was blocked. By immunoelectron microscopy mutant H1 could be directly demonstrated in coated pits. The fraction of wild-type and mutant H1 present in coated pits as determined by immunogold localization correlated well with the respective rates of internalization. Thus, mutation of tyrosine-5 only partially inactivates recognition of H1 for incorporation into coated pits.

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence.

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.

Aminopeptidase N is directly sorted to the apical domain in MDCK cells.

In different epithelial cell types, integral membrane proteins appear to follow different sorting pathways to the apical surface. In hepatocytes, several apical proteins were shown to be transported there indirectly via the basolateral membrane, whereas in MDCK cells a direct sorting pathway from the trans-Golgi-network to the apical membrane has been demonstrated. However, different proteins had been studied in these cells. To compare the sorting of a single protein in both systems, we have expressed aminopeptidase N, which already had been shown to be sorted indirectly in hepatocytes, in transfected MDCK cells. As expected, it was predominantly localized to the apical domain of the plasma membrane. By monitoring the appearance of newly synthesized aminopeptidase N at the apical and basolateral surface, it was found to be directly sorted to the apical domain in MDCK cells, indicating that the sorting pathways are indeed cell type-specific.

[Structure of cholesterol-containing creams]. / Strukturuntersuchungen an cholesterolhaltigen Cremes.

The colloidal structure of a four-component system consisting of cholesterol, fatty alcohol, petrolatum and water was studied. Depending on the variation of temperature, chain length of the fatty alcohol and concentration of all components, creamy systems of w/o type could be prepared, which present different structural features. Liquid phases as well as crystalline and/or liquid crystalline phases participate in the structure of the creams. The liquid phases are represented by water and by liquid hydrocarbons of low molecular weight of petrolatum. Part of the surfactants is dissolved in this latter phase. The liquid crystalline phase is formed by cholesterol and fatty alcohol together. The characterization of the lamellar liquid crystalline phase was performed by polarized light microscopy and small angle x-ray diffraction. The formation of the lamellar liquid crystals is favoured in a certain molar concentration range of cholesterol and fatty alcohol. The liquid crystalline region is largest with tetradecanol. The lamellar liquid crystals swell with water up to a certain limit. This incorporation of water stabilizes the lamellar liquid crystals so that they are stable in a larger temperature range. The crystalline structures form a network consisting of solid hydrocarbons and the eutectic mixture of cholesterol and fatty alcohol. If water is present the surfactants crystallize as hydrates. A surplus of water can be dispersed as droplets mechanically. Referring to maximum capacity of water incorporation, systems with lowest crystalline amounts (only the network of petrolatum) were best. The surfactants should be completely dissolved.