Identification, expression and functional characterization of M(4)L, a muscarinic acetylcholine M(4) receptor splice variant

Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M(4) receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M(4) receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M(4) receptor transcript. The predicted size for the longer two-exon M(4) receptor splice variant with the additional 155 amino acid N-terminal extension, designated M(4)L is 69.7 kDa compared to the 53 kDa canonical single exon M(4) receptor (M(4)S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [(3)H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M(4)L and M(4)S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.