Sequencing of eleven introns in genomic DNA encoding rat glucagon receptor and multiple alternative splicing of its mRNA

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Abstract
We used the PCR (polymerase chain reaction) to amplify fragments of glucagon receptor DNA from genomic DNA. Sequencing of the subcloned fragments demonstrated that genomic DNA encoding the glucagon receptor spans over 12 exons interrupted by 11 introns. The introns were located mainly at the 5′ end and in the core domain of the glucagon receptor CDS totalling 23 kb. Intron positions were similar to the positions of introns in growth hormone‐releasing hormone receptor and parathyroid hormone receptor, two receptors belonging to the same receptor family as the glucagon receptor. This high number of introns might be the cause of the mRNA polymorphism observed at the 5′ end: when PCR was performed on cDNA using primers amplifying the central or 3′ end cDNA fragments, a single band corresponding to the cloned cDNA was observed. In contrast, if primers amplifying cDNA fragments corresponding to nucleotides −8 to 680 of CDS were used, cDNA fragments of approximately 500 bp, 600 bp, 700 bp, 800 bp and 900 bp were specifically and reproducibly amplified. Sequencing of these fragments showed either incomplete intron removal or splicing at alternative positions. Two of these sequenced variants were translatable in putative glucagon receptor variants: (1) unsplicing of intron III (81 bp) gave an additional 27 amino acid sequence after Lys91 in the N‐terminal domain of the receptor. In the liver, where the normal CDS represented about one third of the mRNA molecules, this mRNA variant represented 18% of total mRNA forms; (2) a 21 bp deletion in exon V giving rise to a putative deletion of 7 amino acids in glucagon receptor (Δ64–84 CDS) was also relatively abundant in the liver (10%). The observed polymorphism of the glucagon receptor mRNA may contribute to the regulation of glucagon receptor expression and perhaps to the heterogeneity of these receptors.