SEQUENCE OF THE HUMAN GLYCOGEN-ASSOCIATED REGULATORY SUBUNIT OF TYPE-1 PROTEIN PHOSPHATASE AND ANALYSIS OF ITS CODING REGION AND MESSENGER-RNA LEVEL IN MUSCLE FROM PATIENTS WITH NIDDM

Impaired insulin-stimulated glycogen synthesis of peripheral tissues i s a characteristic feature of many patients with non-insulin-dependent diabetes mellitus (NIDDM) and their first-degree relatives with norma l glucose tolerance, suggesting putative inherited defects in this met abolic pathway. In previous studies, we have failed to reveal mutation s in the coding regions of the muscle-specific glycogen synthase gene and the three genes that encode the catalytic subunits of protein phos phatase 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 syn thesis and the activity of PP1 is decreased in insulin-resistant subje cts, we have now cloned the human G-subunit cDNA to search for abnorma lities in the corresponding gene (designated PPP1R3 in the human genom e nomenclature) in patients with NIDDM. The human cDNA was isolated fr om a skeletal muscle cDNA library and was found to encode a 126-kDa pr otein, which shows 73% amino acid identity to the rabbit PP1 G-subunit . The human G-subunit cDNA from 30 insulin-resistant NIDDM patients wa s analyzed for genetic variations in the G-subunit by using single-str anded conformation polymorphism (SSCP) scanning of reversely transcrib ed 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 s ubstitution from alanine to glutamic acid. The carrier of this mutatio n was characterized by reduced insulin-stimulated nonoxidative glucose metabolism when examined with the euglycemic hyperin-sulinemic clamp. SSCP scanning of the 2584-2844 nucleotide fragment of PP1 G-subunit c DNA from an additional 22 NIDDM patients and 29 control subjects did n ot reveal additional genetic variants. To indirectly screen for abnorm alities in PP1 G-subunit gene regulation, we measured the mRNA level o f the G-subunit in skeletal muscle. However, no difference in the abun dance of mRNA of PP1 G-subunit was found between patients with diabete s and control subjects.