Sequence of the Human Glycogen-Associated Regulatory Subunit of type 1 Protein Phosphatase and Analysis of Its Coding Region and mRNA Level in Muscle From Patients With NIDDM

• Published in: Diabetes (New York, N.Y.) Vol. 43; no. 10; pp. 1234 - 1241
• Main Authors: Hua Chen, Yu, Hansen, Lars, Xiang Chen, Mao, Bjørbæk, Christian, Vestergaard, Henrik, Hansen, Torben, Cohen, Paticia T W, Pedersen, Oluf
• Format: Journal Article
• Language: English
• Published: United States American Diabetes Association 01.10.1994
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Diabetes Mellitus, Type 2 - enzymology; Diabetes Mellitus, Type 2 - genetics; DNA Primers; Female; Genetic aspects; Glucose - metabolism; Glycogen; Glycogen - metabolism; Glycogen synthesis; Humans; Macromolecular Substances; Male; Messenger RNA; Middle Aged; Molecular Sequence Data; Muscles - metabolism; Phosphatases; Phosphoprotein Phosphatases - genetics; Physiological aspects; Polymerase Chain Reaction; Polymorphism, Genetic; Protein Phosphatase 1; Rabbits; Reference Values; RNA, Messenger - metabolism; Sequence Homology, Amino Acid; Synthesis; Type 2 diabetes
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/diab.43.10.1234

Impaired insulin-stimulated glycogen synthesis of peripheral tissues is a characteristic feature of many patients with non-insulin-dependent diabetes mellitus (NIDDM) and their first-degree relatives with normal glucose tolerance, suggesting putative inherited defects in this metabolic pathway. In previous studies, we have failed to reveal mutations in the coding regions of the muscle-specific glycogen synthase gene and the three genes that encode the catalytic subunits of protein phosphatase 1 (PP1) as frequent causes of insulin resistance. Because the glycogen-associated regulatory subunit of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human genome nomenclature) in patients with NIDDM. The human cDNA was isolated from a skeletal muscle cDNA library and was found to encode a 126-kDa protein, which shows 73% amino acid identity to the rabbit PP1 G-subunit. The human G-subunit cDNA from 30 insulin-resistant NIDDM patients was analyzed for genetic variations in the G-subunit by using single-stranded conformation polymorphism (SSCP) scanning of reversely transcribed mRNA. One variant SSCP profile was detected in the region encoding the COOH-terminal part of the PP1 G-subunit in only one NIDDM patient, and subsequent nucleotide sequencing showed a C to A transversion on one allele at base position 2792. This change predicts an amino acid substitution from alanine to glutamic acid. The carrier of this mutation was characterized by reduced insulin-stimulated nonoxidative glucose metabolism when examined with the euglycemic hyperinsulinemic clamp. SSCP scanning of the 2584–2844 nucleotide fragment of PP1 G-subunit cDNA from an additional 22 NIDDM patients and 29 control subjects did not reveal additional genetic variants. To indirectly screen for abnormalities in PP1 G-subunit gene regulation, we measured the mRNA level of the G-subunit in skeletal muscle. However, no difference in the abundance of mRNA of PP1 G-subunit was found between patients with diabetes and control subjects.