The loop between beta-strands beta 2 and beta 3 and its interaction with the N-terminal alpha-helix is essential for biogenesis of alpha 7 nicotinic receptors.
Recently, we have shown that the alpha-helix present at the N-termini of alpha7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking beta-strands beta2 and beta3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant alpha7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (alpha3beta4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric alpha7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the alpha-helix is an important requirement for receptor assembly.
Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Sant Joan d'Alacant, Alicante, Spain.
Manuel
M
Criado
José
J
Mulet
Mar
M
Castillo
Susana
S
Gerber
Salvador
S
Sala
Francisco
F
Sala
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D005260:Female; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050506:Mutant Chimeric Proteins / Q000096:biosynthesis / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
D050506:Mutant Chimeric Proteins; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor