Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsalpha.
Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the alpha subunit of the stimulatory GTP-binding protein of AC (Gsalpha) to stimulate enzyme activity. Concentration-response curves with Gsalpha suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsalpha interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572-F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsalpha-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042-T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsalpha concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042-T1051) in the C2 domain of the enzyme to modulate Gsalpha-elicited stimulation of activity.
Scholich
K
K
Department of Pharmacology, University of Tennessee, Memphis, 874 Union Avenue, Memphis, TN 38163, USA.
Wittpoth
C
C
Barbier
A J
AJ
Mullenix
J B
JB
Patel
T B
TB
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry
D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases