The role for proteoglycans in acetylcholine receptor clustering on cultured muscle.

Persistent Link:
http://hdl.handle.net/10150/187185
Title:
The role for proteoglycans in acetylcholine receptor clustering on cultured muscle.
Author:
Jung, Inhee Mook
Issue Date:
1995
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The present dissertation investigated the possible role of proteoglycans (PGs) in acetycholine receptor (AChR) clustering on cultured C2 myotubes. Analysis of variant muscle cell lines and their hybrid products supported the hypothesis that PGs are required for the clustering of AChRs. Three PG-defective genetic variants derived from the C2 cell line form myotubes but fail to spontaneously cluster AChRs. The three variants show different broad-spectrum defects in glycosaminoglycan (GAG) biosynthesis and are especially deficient in the synthesis of chondroitin sulfate (CS) chain. Formation of heterokaryon myotubes containing nuclei from two different variants spontaneously clustered AChRs and recovered synthesis of GAGs, especially of CS. It strongly suggests that there is a requirement for proper GAG biosynthesis in AChR clustering. Chlorate was found to inhibit both GAG synthesis and the clustering of AChRs in a dose-dependent manner. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild type C2 myotubes, both the frequency of spontaneous AChR clusters and the level of CS were increased. Culture of wild type C2 myotubes in the presence of chondroitinase ABC eliminated CS and prevented the formation of AChR clusters. Treatment with chondroitinase ABC only prevented AChR clustering if begun prior to the formation of spontaneous clusters. This suggests that CS is required in the initiation but not the maintenance of AChR clusters. Agrin-induced AChR clustering was dramatically reduced by digestion of CS. Unlike calcium, however, agrin action on AChR clustering did not affect levels of CS. Also, agrin-induced AChR clustering was restored on S27 myotubes by adding calcium-treated C2 conditioned medium. The IIH6 monoclonal antibody against α-dystroglycan, laminin, and agrin all bound to a specific fraction of C2 cell extracts separated on ion exchange chromatography. Also IIH6, laminin, and agrin affinity-precipitations showed a smeared sulfate labelled band above 120kD which is close in molecular weight to that of $\alpha$-dystroglycan. The band disappeared after chondroitinase ABC treatment. Protease-digested IIH6 immunoprecipitate eluted corresponding to CS. This result strongly suggests that CS is required for agrin activity on AChR clustering.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Cell Biology and Anatomy; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Gordon, Herman

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleThe role for proteoglycans in acetylcholine receptor clustering on cultured muscle.en_US
dc.creatorJung, Inhee Mooken_US
dc.contributor.authorJung, Inhee Mooken_US
dc.date.issued1995en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe present dissertation investigated the possible role of proteoglycans (PGs) in acetycholine receptor (AChR) clustering on cultured C2 myotubes. Analysis of variant muscle cell lines and their hybrid products supported the hypothesis that PGs are required for the clustering of AChRs. Three PG-defective genetic variants derived from the C2 cell line form myotubes but fail to spontaneously cluster AChRs. The three variants show different broad-spectrum defects in glycosaminoglycan (GAG) biosynthesis and are especially deficient in the synthesis of chondroitin sulfate (CS) chain. Formation of heterokaryon myotubes containing nuclei from two different variants spontaneously clustered AChRs and recovered synthesis of GAGs, especially of CS. It strongly suggests that there is a requirement for proper GAG biosynthesis in AChR clustering. Chlorate was found to inhibit both GAG synthesis and the clustering of AChRs in a dose-dependent manner. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild type C2 myotubes, both the frequency of spontaneous AChR clusters and the level of CS were increased. Culture of wild type C2 myotubes in the presence of chondroitinase ABC eliminated CS and prevented the formation of AChR clusters. Treatment with chondroitinase ABC only prevented AChR clustering if begun prior to the formation of spontaneous clusters. This suggests that CS is required in the initiation but not the maintenance of AChR clusters. Agrin-induced AChR clustering was dramatically reduced by digestion of CS. Unlike calcium, however, agrin action on AChR clustering did not affect levels of CS. Also, agrin-induced AChR clustering was restored on S27 myotubes by adding calcium-treated C2 conditioned medium. The IIH6 monoclonal antibody against α-dystroglycan, laminin, and agrin all bound to a specific fraction of C2 cell extracts separated on ion exchange chromatography. Also IIH6, laminin, and agrin affinity-precipitations showed a smeared sulfate labelled band above 120kD which is close in molecular weight to that of $\alpha$-dystroglycan. The band disappeared after chondroitinase ABC treatment. Protease-digested IIH6 immunoprecipitate eluted corresponding to CS. This result strongly suggests that CS is required for agrin activity on AChR clustering.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineCell Biology and Anatomyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairGordon, Hermanen_US
dc.contributor.committeememberBurd, Gail D.en_US
dc.contributor.committeememberErickson, Robert P.en_US
dc.contributor.committeememberRykowski, Maryen_US
dc.contributor.committeememberSt. John, Paul A.en_US
dc.identifier.proquest9534690en_US
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