A mutation of the atrial natriuretic peptide (guanylyl cyclase-A) receptor results in a constitutively hyperactive enzyme

Abstract

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.

Figure 1

Glutamate corresponding to E974 of GC-A is conserved in guanylyl and adenylyl cyclases. The alignment of a sequence within the catalytic domain of GC-A is shown as an example of a membrane guanylyl cyclase isoform, the α₁ and β₁ subunits of the soluble isoform, and the first and second cytoplasmic domain (C₁ and C₂) of adenylyl cyclase IV. The corresponding amino acids are 957–977 (GC-A), 590–610 (α₁), 537–557 (β₁), 383–403 (adenylyl cyclase IV, C₁), and 990-1010 (adenylyl cyclase IV, C₂).

Figure 2

Activity of GC-A and of GC-A E974A in membranes of transfected cells. Guanylyl cyclase activity was estimated in the presence of Mn²⁺ containing 1% Triton X-100. (Inset) Western blots demonstrating equivalent expression of GC-A and GC-A E974A.

Figure 3

Effects of ANP on cGMP accumulation in cells or on guanylyl cyclase activity in broken-cell preparations. (A) ANP (1 μM) or no ANP was added to COS-7 cells transfected with pCMV5, GC-A, or GC-A E974A. (B) Membranes prepared from transfected COS-7 cells were assayed for guanylyl cyclase activity in the presence of Mg²⁺ and in the presence or absence of ANP/ATP.

Figure 4

Effects of HCAT or of HCAT E974A on cellular cGMP concentrations or guanylyl cyclase activity. (A) COS-7 cells were transfected with pCMV5, HCAT, or HCAT E974A and cGMP concentrations were measured. (B) The soluble fraction of transfected COS-7 cells was collected after homogenization and the activity of guanylyl cyclase estimated. (Inset) Western blots demonstrating equivalent expression of HCAT and E974A HCAT.

Figure 5

Guanylyl cyclase activity as a function of MnGTP concentration. Guanylyl cyclase activity was estimated as described in the text and plotted in double reciprocal form.