Characterization of soluble forms of nonchimeric type V adenylyl cyclases.
Type V adenylyl cyclase (ACV) belongs to the family of Ca2+-inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the alpha subunit of the stimulatory G protein Gs (G(s alpha)) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the alpha subunit of the inhibitory G protein Gi (G(i alpha)) inhibited all forms of the enzyme, indicating that the regions for G(s alpha) and G(i alpha) interaction are preserved in the soluble forms. Ca2+ inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G(s alpha) and forskolin obliterated the Ca2+-mediated inhibition of the full-length and C1-C2 forms of ACV. In 45Ca2+ overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca2+. Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound 45Ca2+. To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G(s alpha) and G(i alpha) is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca2+ and inhibition of enzyme activity.
Scholich
K
K
Department of Pharmacology, University of Tennessee, Memphis 38163, USA.
Barbier
A J
AJ
Mullenix
J B
JB
Patel
T B
TB
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera
D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002118:Calcium