Evidence for domain organization within the 61-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain

Abstract

The complete amino acid sequence of the 61-kDa calmodulin-dependent, cyclic nucleotide phosphodiesterase (CaM-PDE) from bovine brain has been determined. The native protein is a homodimer of N alpha-acetylated, 529-residue polypeptide chains, each of which has a calculated molecular weight of 60,755. The structural organization of this CaM-PDE has been investigated with use of limited proteolysis and synthetic peptide analogues. A site capable of interacting with CaM has been identified, and the position of the catalytic domain has been mapped. A fully active, CaM-independent fragment (Mr = 36,000), produced by limited tryptic cleavage in the absence of CaM, represents a functional catalytic domain. N-Terminal sequence and size indicate that this 36-kDa fragment is comprised of residues 136 to approximately 450 of the CaM-PDE. This catalytic domain encompasses a approximately 250 residue sequence that is conserved among PDE isozymes of diverse size, phylogeny, and function. CaM-PDE and its PDE homologues comprise a unique family of proteins, each having a catalytic domain that evolved from a common progenitor. A search of the sequence for potential CaM-binding sites revealed only one 15-residue segment with both a net positive charge and the ability to form an amphiphilic alpha-helix. Peptide analogues that include this amphiphilic segment were synthesized. Each was found to inhibit the CaM-dependent activation of the enzyme and to bind directly to CaM with high affinity in a calcium-dependent manner. This site is among the sequences cleaved from a 45-kDa chymotryptic fragment that has the complete catalytic domain but no longer binds CaM. These results indicate that residues located between position 23 and 41 of the native enzyme contribute significantly to the binding of CaM although the involvement of residues from additional sites is not excluded.

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

“…2A, which shows that the heat effects during initial injections are comparable and are followed by a quick disappearance of heat effects in later injections, indicating that saturation of Ca (Table I). This includes the PDE peptide, which in a previous study had been observed to bind Ca 2ϩ -CaM with a stoichiometry of 2 peptides per CaM molecule (53). The results here, as well as those of another study (22), however, strongly suggest a 1:1 mode of binding for this PDE peptide to CaM.…”

Water Channels and Zymogen Granules in Salivary Glands

“…PDE families have different responses to inhibitors. PDE1s are stimulated by Ca 2+ / CaM activity (134,135), and PDE4 regulates cAMP signaling by its PKA-mediated phosphorylation (136 -138). PKA is a major target of cAMP-regulated exocytosis in exocrine cells.…”

Expression of an Active, Monomeric Catalytic Domain of the cGMP-binding cGMP-specific Phosphodiesterase (PDE5)

“…Mammalian PDEs form a superfamily of enzymes comprised of 10 families of PDEs which differ in amino acid sequences (2, 3, 9 -11), substrate specificities, inhibitor sensitivities, modes of regulation, and tissue distribution. PDEs are chimeric proteins (12)(13)(14)(15) which contain specific domains that provide for cyclic nucleotide hydrolysis, interaction with allosteric/regulatory molecules, autoinhibition, subcellular localization, and perhaps for other functions as well (12,16,17). For mammalian PDEs, the regulatory domains appear to reside mainly in the amino-terminal portions of the proteins.…”