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Rapsyn interacts with the muscle acetylcholine receptor via alpha-helical domains in the alpha, beta, and epsilon subunit intracellular loops.

ABSTRACT: At the developing vertebrate neuromuscular junction, the acetylcholine receptor becomes aggregated at high density in the postsynaptic muscle membrane. Receptor localization is regulated by the motoneuron-derived factor, agrin, and requires an intracellular, scaffolding protein called rapsyn. However, it remains unclear where rapsyn binds on the acetylcholine receptor and how their interaction is regulated. In this study, we identified rapsyn's binding site on the acetylcholine receptor using chimeric constructs where the intracellular domain of CD4 was substituted for the major intracellular loop of each mouse acetylcholine receptor subunit. When expressed in heterologous cells, we found that rapsyn clustered and cytoskeletally anchored CD4-alpha, beta and epsilon subunit loops but not CD4-delta loop. Rapsyn-mediated clustering and anchoring was highest for beta loop, followed by epsilon and alpha, suggesting that rapsyn interacts with the loops with different affinities. Moreover, by making deletions within the beta subunit intracellular loop, we show that rapsyn interacts with the alpha-helical region, a secondary structural motif present in the carboxyl terminal portion of the subunit loops. When expressed in muscle cells, rapsyn co-immunoprecipitated together with a CD4-alpha helical region chimera, independent of agrin signaling. Together, these findings demonstrate that rapsyn interacts with the acetylcholine receptor via an alpha-helical structural motif conserved between the alpha, beta and epsilon subunits. Binding at this site likely mediates the critical rapsyn interaction involved in localizing the acetylcholine receptor at the neuromuscular junction.

SUBMITTER: Lee Y

PROVIDER: S-EPMC2728176 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Rapsyn interacts with the muscle acetylcholine receptor via alpha-helical domains in the alpha, beta, and epsilon subunit intracellular loops.

Lee Y Y Rudell J J Ferns M M

Neuroscience 20090529 1

At the developing vertebrate neuromuscular junction, the acetylcholine receptor becomes aggregated at high density in the postsynaptic muscle membrane. Receptor localization is regulated by the motoneuron-derived factor, agrin, and requires an intracellular, scaffolding protein called rapsyn. However, it remains unclear where rapsyn binds on the acetylcholine receptor and how their interaction is regulated. In this study, we identified rapsyn's binding site on the acetylcholine receptor using ch ...[more]

PMID: 19482062

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Project description:Loops connecting the transmembrane (TM) alpha-helices in membrane proteins are expected to affect the structural organization of the thereby connected helices and the helical bundles as a whole. This effect, which has been largely ignored previously, is studied here by analyzing the x-ray structures of 41 alpha-helical membrane proteins. First we define the loop flexibility ratio, R, and find that 53% of the loops are stretched, where a stretched loop constrains the distance between the two connected helices. The significance of this constraining effect is supported by experiments carried out with bacteriorhodopsin and rhodopsin, in which cutting or eliminating their (predominately stretched) loops has led to a decrease in protein stability, and for rhodopsin, in most cases, also to the destruction of the structure. We show that for nonstretched loops in the extramembranous regions, the fraction of hydrophobic residues is comparable to that for soluble proteins; furthermore (as is also the case for soluble proteins), the hydrophobic residues in these regions are preferentially buried. This is expected to lead to the compact structural organization of the loops, which is transferred to the TM helices, causing them to assemble. We argue that a soluble protein complexed with a membrane protein similarly promotes compactness; other properties of such complexes are also studied. We calculate complementary attractive interactions between helices, including hydrogen bonds and van der Waals interactions of sequential motifs, such as GXXXG. The relative and combined effects of all these factors on the association of the TM helices are discussed and protein structures with only a few of these factors are analyzed. Our study emphasizes the need for classifying membrane proteins into groups according to structural organization. This classification should be considered when procedures for structural analysis or prediction are developed and applied. Detailed analysis of each structure is provided at http://flan.blm.cs.cmu.edu/memloop/

Dataset Information

Rapsyn interacts with the muscle acetylcholine receptor via alpha-helical domains in the alpha, beta, and epsilon subunit intracellular loops.

Publications

Rapsyn interacts with the muscle acetylcholine receptor via alpha-helical domains in the alpha, beta, and epsilon subunit intracellular loops.

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