Determinants in the β and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.
The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.
From the Departments of Physiology and Membrane Biology.
Jolene Chang
JC
Rudell
Lucia S
LS
Borges
John B
JB
Rudell
Kenneth A
KA
Beck
Michael J
MJ
Ferns
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D020125:Mutation, Missense; D021382:Protein Sorting Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*
D009124:Muscle Proteins; D021382:Protein Sorting Signals; D011978:Receptors, Nicotinic
Golgi; Neuromuscular Junction; Nicotinic Acetylcholine Receptors; Protein Motifs; Skeletal Muscle; Trafficking