A mutation of the atrial natriuretic peptide (guanylyl cyclase-A) receptor results in a constitutively hyperactive enzyme.
Mutation of an invariant glutamate residue found within the catalytic domain of guanylyl cyclases resulted in a dramatic 14-fold increase in the activity of the guanylyl cyclase-A receptor. Even in the presence of Mn2+/Triton X-100, a treatment previously thought to yield hormone-independent and maximum cyclase activity, the mutant enzyme remained 7-fold more active; to our knowledge, this is the first example of a protein modification or of an added agent that significantly increases cyclase activity in the presence of Mn2+/Triton X-100. Intracellular concentrations of cGMP in cells expressing the mutant (E974A) cyclase were only marginally elevated by the addition of atrial natriuretic peptide, and in broken-cell preparations, the mutant enzyme also was relatively insensitive to ligand/regulatory nucleotide. The marked increase in cyclase activity was not due to a relief of protein kinase domain inhibition, since the point mutation caused 7- to 13-fold elevations in guanylyl cyclase-A activity when the protein kinase homology domain was deleted. The E974A mutation also altered the kinetics from positive cooperative to linear with respect to MnGTP, suggesting disruption of subunit-subunit interactions. Thus, a single point mutation within the catalytic domain of a guanylyl cyclase results in a constitutively hyperactive enzyme that is independent of protein kinase domain regulation.
Wedel
B J
BJ
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Foster
D C
DC
Miller
D E
DE
Garbers
D L
DL
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation; D005971:Glutamates / Q000737:chemistry; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D005971:Glutamates; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D006152:Cyclic GMP