Identification of the retinal cyclic GMP phosphodiesterase inhibitory gamma-subunit interaction sites on the catalytic alpha-subunit.
Retinal rod outer segment phosphodiesterase (PDE) consists of two similar catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). A trypsin-activated soluble PDE exhibiting the ability to be reinhibited by PDE gamma was shown by peptide antisera to retain both N and C termini. Synthetic peptides corresponding to residues 16-30, 78-90, 389-403, and 535-563 of PDE alpha used in a PDE activity assay with trypsin-activated PDE partially prevented inhibition by exogenous PDE gamma; however, only competitions by peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90) were concentration-dependent below 100 nmol of peptide. Binding studies using radio-immunoassays and PDE alpha peptides confirmed that peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90, respectively) were able to bind PDE gamma. Additionally, peptides corresponding to the PDE alpha region 453-534 bound PDE gamma in the binding assay. This suggests that several regions on PDE alpha interact with the PDE gamma inhibitor. While some regions may be involved in binding to PDE gamma, other sites may be involved in PDE gamma inhibition of catalytic activity. Our results suggest that the major regions of PDE alpha that interact with PDE gamma reside within the N terminus (16-30 and 78-90), with weaker interaction regions within or near the hypothesized catalytic domain (453-563). Sequence analysis of three retinal phosphodiesterases (rod outer segment alpha, beta, and cone outer segment alpha') revealed the highest region of dissimilarity in the N and C termini.
Oppert
B
B
Department of Biochemistry, Kansas State University, Manhattan 66506.
Cunnick
J M
JM
Hurt
D
D
Takemoto
D J
DJ
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D008969:Molecular Sequence Data; D011863:Radioimmunoassay; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D016415:Sequence Alignment; D012995:Solubility; D014357:Trypsin / Q000378:metabolism
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin