Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid.

Abstract

p24 is a thyroid protein (Mr 24,000) identified by two‐dimensional gel electrophoresis on the basis that its synthesis and phosphorylation are up‐regulated by thyrotropin and cyclic AMP agonists. p24 cDNA was cloned from a lambda gt11 cDNA library using a polyclonal antibody raised against the protein recovered from a Western blot spot. The encoded polypeptide (189 residues) displays a putative target‐site for phosphorylation by cyclic AMP‐dependent protein kinase and belongs to the superfamily of proteins binding Ca2+ through ‘EF hand’ domains. It presents four such domains of which two agree closely with the consensus. The ability of p24 to bind Ca2+ has been directly confirmed on Western blots. p24 was detected in many tissues including the salivary glands, the lung and the brain. The ubiquitous nature of p24, together with its regulatory and sequence characteristics suggest that it constitutes an important target common to the cyclic AMP and Ca2+‐phosphatidylinositol cascades.

A Novel EF‐Hand Ca2+‐Binding Protein from Abdominal Muscle of Crustaceans with Similarity to Calcyphosine from Dog Thyroidea

“…Partial sequence analysis (137 residues) of CCBP-23 from the lobster, Homarus americanus, showed a sequence identity of 74% with the crayfish protein. Homology searches revealed a 44% sequence identity of CCBP-23 from crayfish to calcyphosine, a Ca2+-binding protein from dog thyroidea (Lefort et al, 1989). Although CCBP-23 also shows a 44% identity to R2D5 antigen (Nemoto et al, 1993), we believe that both proteins represent two distinct subgroups within the family of EF-hand proteins.…”

“…Assuming a similar efficiency of the purification procedure for CCBP-23 as for calmodulin, whose low abundance in striated muscle tissues is known, Table 1. Protein sequences were compared with the sequence of calcyphosine (CalphosfC) deduced from its cDNA (Lefort et al, 1989). Dots indicate sequence identity.…”

“…A search in the SwissProt and the EMBL data banks revealed a remarkable similarity of CCBP-23 to calcyphosine, a protein from dog thyroidea (Lefort et al, 1989), also present in rat brain (Mailleux et al, 1993). The canine calcyphosine cDNA revealed a four-domain EF-hand protein (189 residues, 21 104 Da) with a sequence identity of 31 % to bovine calmodulin as the most closely related protein.…”

“…This is nearly twice as long as in a typical four domain EF-hand protein such as calmodulin, and therefore represents a further peculiarity of CCBP-23 and calcyphosine. The similarity between the two proteins furthermore includes an identical PI (5.4) and their ability to bind in a Ca'+-dependent manner to phenyl-Sepharose (Lefort et al, 1989).…”

“…We name these novel proteins . Both show similarity to the EF-hand protein calcyphosine from dog thyroidea (Lefort et al, 1989) and the R2D5 antigen (Nemoto et al, 1993), but not to any other known sequence.…”