Rapsyn Escorts the Nicotinic Acetylcholine Receptor Along the Exocytic Pathway via Association with Lipid Rafts

Abstract

The 43 kDa receptor-associated protein rapsyn is a myristoylated peripheral protein that plays a central role in nicotinic acetylcholine receptor (AChR) clustering at the neuromuscular junction. In a previous study, we demonstrated that rapsyn is specifically cotransported with AChR via post-Golgi vesicles targeted to the innervated surface of the Torpedo electrocyte (Marchand et al., 2000). In the present study, to further elucidate the mechanisms for sorting and assembly of postsynaptic proteins, we analyzed the dynamics of the intracellular trafficking of fluorescently labeled rapsyn in the transient-expressing COS-7 cell system. Our approach was based on fluorescence, time-lapse imaging, and immunoelectron microscopies, as well as biochemical analyses. We report that newly synthesized rapsyn associates with the trans-Golgi network compartment and traffics via vesiculotubular organelles toward the cell surface of COS-7 cells. The targeting of rapsyn organelles appeared to be mediated by a microtubule-dependent transport. Using cotransfection experiments of rapsyn and AChR, we observed that these two molecules codistribute within distal exocytic routes and at the plasma membrane. Triton X-100 extraction on ice and flotation gradient centrifugation demonstrated that rapsyn and AChR are recovered in low-density fractions enriched in two rafts markers: caveolin-1 and flotillin-1. We propose that sorting and targeting of these two companion molecules are mediated by association with cholesterol-sphingolipid-enriched raft microdomains. Collectively, these data highlight rapsyn as an itinerant vesicular protein that may play a dynamic role in the sorting and targeting of its companion receptor to the postsynaptic membrane. These data also raise the interesting hypothesis of the participation of the raft machinery in the targeting of signaling molecules to synaptic sites.

Lipid Rafts Serve as a Signaling Platform for Nicotinic Acetylcholine Receptor Clustering

“…Rapsyn is constitutively localized in lipid rafts (Fig. 8 A), presumably via myristoylation at the N terminus (Marchand et al, 2002), whereas the AChR is present in nonraft microdomains in naive muscle cells (Fig. 1 A).…”

“…Lipid rafts were prepared as described previously (Marchand et al, 2002). Briefly, C2C12 myotubes from a 60 mm dish, either control or treated, were collected in PBS and resuspended in 2 ml of lysis buffer containing 25 mM 2-[N-morpholino] ethanesulfonic acid, pH 6.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, 10 g/ml aprotinin, 1 g/ml pepstatin A, and 1 g/ml leupeptin.…”

“…There is a consensus myristoylation site in the N terminal of the rapsyn protein. Rapsyn is enriched in the lipid raft fraction when expressed in human embryonic kidney 293 cells (Marchand et al, 2002). However, whether the endogenous rapsyn in muscle cells partition in lipid rafts and, if so, whether it is regulated by agrin remained unclear.…”

Acetylcholine Receptors Direct Rapsyn Clusters to the Neuromuscular Synapse in Zebrafish

“…Instead, receptors might be required for the formation of a larger synaptic complex that serves as a prerequisite for anchoring. A different role for the AChR in rapsyn navigation is suggested on the bases of recent studies showing that rapsyn and receptor are cotransported and inserted into the plasma membrane (Marchand et al, 2000(Marchand et al, , 2002. The lack of receptor in the complex could abrogate insertion and lead to ineffective assembly at the subsynaptic sites.…”

Molecular mechanisms of acetylcholine receptor–lipid interactions: from model membranes to human biology

“…Both muscle-type and neuronal nAChRs require lipid rafts for trafficking to the plasma membrane, with the raft-forming lipids cholesterol, sphingolipids, and ceramides being important Gallegos et al 2008;Baier et al 2010;Bruses et al 2001;Marchand et al 2002). Both the clustering of nAChRs on the plasma membrane and the internalization of cytoplasmic membrane-located nAChRs are raft dependent.…”