Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase IIβ

D. B. Bregman, A. H. Hirsch, C. S. Rubin

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Abstract

In mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase IIβ with anchoring proteins that bind the regulatory subunit (RIIβ) of the enzyme. Previously, an RIIβ-binding domain was characterized in a large (M(r) ~ 150,000) candidate anchor protein, rat brain P150 (Bregman, D. B., Bhattacharyya, N., and Rubin, C. S. (1989) J. Biol. Chem. 264, 4648-4656). RIIβ-binding proteins with M(r) values of 65,000-80,000 were detected in the brains of other species. Since little was known about the structural features of these lower M(r) proteins, we undertook the characterization of bovine brain P75 as a prototype. A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a λgt11 expression library with 32P-RIIβ. The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli. A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised. Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain. The sequence of 117 amino acid residues at the C terminus of P75 contains the RIIβ-binding site and is 80% identical to the corresponding region of P150. In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RIIβ-binding proteins are related, but distinct proteins. P75 is not homologous to microtubule-associated protein 2, an RIIα-selective binding protein, or any other previously studied proteins. C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RIIβ.

Original languageEnglish (US)
Pages (from-to)7207-7213
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number11
StatePublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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