Nucleoside diphosphate kinase-mediated activation of heterotrimeric G proteins.
Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to receptor independent G protein activation. Apparently, the NDPK B isoform forms complexes with Gbetagamma dimers and thereby phosphorylates His266 in Gbeta1 subunits. Phosphorylated His266 mediates G protein activation by a transfer of the high energetic phosphate onto GDP, thus leading to de novo synthesis of GTP. Moreover, it has been demonstrated that the sarcolemmal content of NDPK isoforms is increased in hearts with terminal congestive heart failure leading to enhanced G protein activation. Similar data were reported in a rat model for beta-adrenoceptor-induced cardiac hypertrophy. We therefore describe in this chapter several methods which can be used for analysis of NDPK mediated G protein activation: (1) The quantification of NDPK isoforms in highly purified cardiac sarcolemmal membranes, (2) the enrichment of the NDPK B/Gbetagamma-complex from preparations of the retinal G protein transducin, (3) the analysis of the enhanced NDPK activated and high energy phosphate transfer in a neonatal rat cardiac myocyte derived cell line stably overexpressing NDPK (H10 cells), and (4) the increased activation of adenylyl cyclase by the enhanced receptor-independent activation of the stimulatory G protein alpha subunit in these cells.
Institut für Pharmakologie und Toxikologie, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Germany.
Susanne
S
Lutz
Hans-Jörg
HJ
Hippe
Feraydoon
F
Niroomand
Thomas
T
Wieland
D015720:5'-Nucleotidase / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D009206:Myocardium / Q000166:cytology / Q000201:enzymology; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D012374:Rod Cell Outer Segment / Q000737:chemistry; D012508:Sarcolemma / Q000737:chemistry / Q000201:enzymology; D015293:Transducin / Q000737:chemistry / Q000378:metabolism
D044385:GTP-Binding Protein alpha Subunits; D007527:Isoenzymes; D046911:Macromolecular Substances; D021122:Protein Subunits; D000242:Cyclic AMP; D009701:Nucleoside-Diphosphate Kinase; D015720:5'-Nucleotidase; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin