Binding proteins for the regulatory subunit (RII-B) of brain cAMP-dependent protein kinase II: isolation and initial characterization of cDNA clones
In mammalian brain several proteins bind RII-B with high affinity. An example is P75, which co-purifies with RII-B and also complexes CaS -calmodulin. Thus, RII-B binding proteins (RBPs) might play a role in integrating the CaS and cAMP signalling pathways in the CNS. In order to study the structure and function of these polypeptides they have isolated cloned cDNAs for RBPs by screening brain lambdagt11 expression libraries using a functional assay: the binding of TSP-labeled RII to fusion proteins produced by recombinants expressing RII binding domains. Inserts from rat brain recombinant clones lambda7B and lambda10B both hybridize to a brain mRNA of 7000 nucleotides. Northern gel analyses indicate that the putative RBP mRNA is also expressed in lung, but not in several other tissues. The lambda7B insert was subcloned into the expression plasmid pINIA. A 50 kDa high affinity RII-B binding polypeptide accumulated in E. coli transformed with pINIA-7B. Two RBP cDNAs (lambda77, lambda100A) have been retrieved from a bovine lambdagt 11 library using a monoclonal antibody directed against P75 and the binding assay respectively. On Southern blots the insert from lambda100A hybridizes to the cDNA insert from clones lambda77, suggesting that lambda 77 cDNA might contain sequences coding for both an RII binding domain and a P75 epitope. The bovine lambda100A insert also hybridizes with the rat lambda7B clone indicating that an RII binding domain is conserved in the two species.
- Research Organization:
- Albert Einstein College of Medicine, Bronx, NY
- OSTI ID:
- 6359018
- Report Number(s):
- CONF-870644-; TRN: 87-028704
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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