Interactions of the Rapsyn RING-H2 Domain with Dystroglycan

• Published in: The Journal of biological chemistry 2001-07, Vol.276 (27), p.24911-24917
• Main Authors: Bartoli, M, Ramarao, M K, Cohen, J B
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Cell Line; Conserved Sequence; Cytoskeletal Proteins - metabolism; Dystroglycans; Humans; Membrane Glycoproteins - metabolism; Membrane Proteins - metabolism; Muscle Proteins - chemistry; Muscle Proteins - metabolism; Peptide Mapping; Protein Binding; Receptors, Nicotinic - metabolism; Structure-Activity Relationship; Utrophin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M103258200

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 β-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 β-subunit fragment, the β-dystroglycan cytoplasmic domain, or rapsyn domains. The rapsyn coiled-coil domain interacts with the nAChR β-subunit cytoplasmic domain, but not with itself, other rapsyn domains, or β-dystroglycan. The RING-H2 domain interacts only with the β-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.