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Amino acid residues in transmembrane domain 1 and in the membrane proximal C‐tail of muscarinic M 1 receptors are necessary for transport‐competent folding
Author(s) -
Sawyer Gregory William,
Shults Crystal Alane
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.945.4
Subject(s) - receptor , transmembrane domain , muscarinic acetylcholine receptor , microbiology and biotechnology , mutant , chemistry , g protein coupled receptor , wild type , biology , biochemistry , gene
The principle goal of this investigation was to identify the functional role of a conserved C‐terminal tail motif, 423‐F(X) 6 LL‐431, in muscarinic M 1 receptors. Point mutations were introduced into this motif, and at the base of transmembrane spanning domain 1 (TM1), using site‐directed mutagenesis. Wild‐type and mutant M 1 receptors were transiently expressed in CHO cells and the amount of receptor expressed on the plasma membrane was determined using intact, whole cell [ 3 H]‐ N ‐methylscopolamine ([ 3 H]NMS) binding assays. Mutation of Leu pair 430‐LL‐431 in the F(X) 6 LL motif to an Ala pair (hM 1LL430‐431 ) caused a 92% reduction in the plasma membrane expression of M 1 receptors. Similarly, mutation of 46‐VL‐47 at the base of TM1 to 46‐AA‐47 (hM 1VL46‐47 ) caused a 98% reduction in plasma membrane expression. The plasma membrane expression of hM 1LL430‐431 and hM 1VL46‐47 receptors increased 5.3‐ and 18.2‐fold, respectively, when incubated with atropine (0.1 μM) for 18 h. Unlike GFP‐tagged M 1 receptors, GFP‐tagged M 1LL430‐431 receptors colocalized extensively with the ER marker DsRed‐ER. When incubated with atropine (0.1 mM) for 18 h, GFP‐tagged M 1LL430‐431 receptors acquired a cellular localization consistent with that of wild‐type receptors (i.e., expressed on the plasma membrane). Collectively, these data suggest that 430‐LL‐431 and 46‐VL‐47 are necessary for transport‐competent folding of M 1 receptors.