Dominant negative mutations of the guanylyl cyclase-A receptor. Extracellular domain deletion and catalytic domain point mutations.
Guanylyl cyclase-A (GC-A), a receptor for A-type natriuretic peptide (ANP), contains an extracellular ligand-binding domain, a single transmembrane domain, and intracellular protein kinase-like and cyclase catalytic domains. Expression of the putative cyclase catalytic region (HCAT) resulted in the formation of an active enzyme that migrated as a homodimer on gel filtration columns; treatment with sodium trichloroacetate caused dissociation of the dimer and a loss of cyclase activity. Co-transfection of HCAT and full-length GC-A led to elevated basal intact cell cGMP concentrations and increased cell homogenate guanylyl cyclase activity. However, atrial natriuretic peptide-induced elevations of cGMP and cyclase activity were inhibited by the introduction of HCAT. Alanine scanning mutagenesis of highly conserved residues within HCAT identified one mutation (D893A) that destroyed enzyme activity but not the ability of the mutant subunit to form homodimers. The mutant subunit inhibited the cyclase activity of wild-type HCAT (approximately 70%) as well as that of full-length GC-A (approximately 85%) in co-expression studies where the amount of wild-type HCAT or full-length GC-A was not altered. Unlike co-transfection with wild-type HCAT, co-transfection of HCA-TD893A and GC-A did not result in elevated basal intact cell cGMP concentrations. For the first time we describe deletion and point mutations within the plasma membrane family of guanylyl cyclase receptors that result in the formation of effective dominant negative proteins.
Thompson
D K
DK
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235-9050.
Garbers
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D005799:Genes, Dominant; D006162:Guanylate Cyclase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion
D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A