Control of AMP deaminase 1 binding to myosin heavy chain

Abstract

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178–333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660–674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8–12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

L-Cysteine Prevents Oxidation-Induced Block of the Cardiac Na+ Channel Via Interaction With Heart-Specific Cysteinyl Residues in the P-Loop Region.

“…COS7 cells grown on glass cover slips were cotransfected with both wild type and mutant hSkM1, or hH1 cDNA and green fluorescence protein cDNA (Invitrogen). 13 After 48 h, whole cell patch-clamp studies were performed using the cells that expressed the green fluorescence protein.…”

A Novel Bipartite Intronic Splicing Enhancer Promotes the Inclusion of a Mini-exon in the AMP Deaminase 1 Gene

“…This enzyme is also influenced allosterically by cellular levels of purine nucleotides, through a binding site near its amino terminus (12, 24 -28). We have shown that the four-amino acid peptide encoded by exon 2 influences both of these properties in a fiber type-dependent fashion (19). In adult, fast twitch, glycolytic myofibers, the sensitivity of AMPD to cellular ATP/GTP levels is altered significantly by the presence of the exon 2 domain (14,16,19).…”

“…We have shown that the four-amino acid peptide encoded by exon 2 influences both of these properties in a fiber type-dependent fashion (19). In adult, fast twitch, glycolytic myofibers, the sensitivity of AMPD to cellular ATP/GTP levels is altered significantly by the presence of the exon 2 domain (14,16,19). In the resting state, ATP levels in the myocyte are relatively high, and a purine nucleotide binding site near the carboxyl terminus of AMPD is occupied.…”

“…In the resting state, ATP levels in the myocyte are relatively high, and a purine nucleotide binding site near the carboxyl terminus of AMPD is occupied. The exon 2-minus isoform of AMPD, which predominates in slow twitch, oxidative myofibers, can bind to myosin heavy chain and become activated in the presence of relatively high concentrations of ATP (12,19). Thus, the essential role of AMPD in generating both fumarate (for use as a citric acid cycle intermediate) and IMP (from the deamination of AMP) can be retained in both fiber types, at least in part, by regulating the alternative splicing of exon 2.…”

“…This 12-base mini-exon is largely, but not exclusively, retained in adult myotubes (17). Recent data suggest a role for the exon 2-encoded peptide in altering the allosteric responsiveness of AMPD to intracellular ATP levels, clearly important for the homeostasis of muscle (19). A recently discovered genetic lesion, a C-T transition that results in the introduction of a nonsense mutation at the end of exon 2, is partly responsible for human AMPD deficiency.…”