Identification and characterization of a novel A-kinase-anchoring protein (AKAP120) from rabbit gastric parietal cells.
The type-II cAMP-dependent protein kinase (A-Kinase) partitions primarily into the particulate fraction in gastric parietal cells. Localization of this kinase to particular subcellular domains is mediated through the binding of the regulatory subunit (RII) dimer to A-Kinase-anchoring proteins (AKAPs). Using a [32P]RII overlay assay, we have screened a rabbit gastric parietal cell cDNA library and have isolated a single RII-binding protein clone. Sequence analysis revealed an open reading frame coding for 1022 amino acids (AKAP120). Recombinant fragments of the full-length clone were prepared and the RII-binding region mapped to an area between amino acids 489 and 549. This area contained a putative alpha-helical RII-binding region between amino acids 503 and 516. Incubation of [32P]RII with a synthetic peptide of AKAP120-(489-522) completely inhibited the binding of [32P]RII to the recombinant AKAP120 fragments that demonstrated RII binding. In vitro RII-binding affinity studies indicated a high-affinity interaction between AKAP120 and RII with a Kapp between 50 and 120 nM for the three recombinant fragments that bound [32P]RII. RNase-protection analysis revealed that AKAP120 is a widely distributed protein, with the highest levels of mRNA observed in gastric fundus. The presence of this novel high-affinity AKAP in gastric parietal cells suggests that it may regulate RII subcellular sequestration in this cell type.
Dransfield
D T
DT
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-3175, USA.
Yeh
J L
JL
Bradford
A J
AJ
Goldenring
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D017421:Sequence Analysis
D002352:Carrier Proteins; D018076:DNA, Complementary; D011506:Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases