Abstract
Rapsyn, a peripheral membrane protein of skeletal muscle, is necessary for the formation of the highly organized structure of the vertebrate neuromuscular junction. For mice lacking rapsyn, there is a failure of postsynaptic specialization characterized by an absence of nicotinic acetylcholine receptors (nAChRs) and other integral and peripheral membrane proteins such as beta-dystroglycan and utrophin. Dystroglycan is necessary for the formation of the mature neuromuscular junction and has been shown to interact directly with rapsyn. Previous studies with rapsyn fragments and mutants, expressed in 293T cells along with nAChRs, establish that the rapsyn tetratricopeptide repeat (TPR) domain is involved in self-association and its coiled-coil domain is necessary for nAChR clustering. The function of the rapsyn RING-H2 domain, which is not necessary for rapsyn self-association or nAChR clustering, is unknown. To further characterize these domains, we have used a yeast two-hybrid assay to test for interactions at the plasma membrane between rapsyn domains and a nAChR beta-subunit fragment, the beta-dystroglycan cytoplasmic domain, or rapsyn domains. The rapsyn coiled-coil domain interacts with the nAChR beta-subunit cytoplasmic domain, but not with itself, other rapsyn domains, or beta-dystroglycan. The RING-H2 domain interacts only with the beta-dystroglycan cytoplasmic domain. Furthermore, when expressed in 293T cells, a rapsyn construct containing as few as two TPRs and the RING-H2 domain self-associates and clusters dystroglycan, but not nAChRs. These results emphasize the modular character of the rapsyn structural domains.
Highlights
Plasma membrane, but they are not clustered [4]
Previous studies with rapsyn fragments and mutants, expressed in 293T cells along with nicotinic acetylcholine receptors (nAChRs), establish that the rapsyn tetratricopeptide repeat (TPR) domain is involved in self-association and its coiled-coil domain is necessary for nAChR clustering
The expression of human Son of Sevenless (hSos), the mammalian homologue of cdc25, is able to complement the cdc25 mutation only if it can be localized to the plasma membrane. hSos membrane localization can be accomplished if it is expressed as a hybrid protein fused to a bait protein that interacts with a partner which is localized at the plasma membrane
Summary
All restriction enzymes were purchased from New England Biolabs. T4 DNA ligase and oligonucleotides were obtained from Life Technologies, Inc. Polymerase chain reactions (PCR) were carried out in 100 l using 20 ng of the templates described below, 50 –100 pmol of each primer, 250 M each dNTP, and 5 units of Pfu DNA polymerase (Stratagene) for 30 cycles at 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min for 30 cycles. All constructs were tested both by restriction enzyme analysis and by sequencing across the full length of the inserted fragments. Yeast cdc25H Saccharomyces cerevisiae as well as the vectors pSos and pMyr were purchased from Stratagene. The vector p5ЈSos [19] was provided by Dr A. The plasmids pCDNA3-dystroglycan (mouse) and pCDNA3-dystroglycan (rabbit) were provided by Drs S.
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