Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 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.