ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Amino acid sequence of ARPP-21B from bovine caudate nucleus

J Neurosci. 1989 Oct;9(10):3631-7. doi: 10.1523/JNEUROSCI.09-10-03631.1989.

Abstract

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS-PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of the rat CNS. It has recently been purified to homogeneity from bovine caudate nucleus and characterized (Hemmings and Greengard, 1989). ARPP-21 is isolated as 2 isoforms, ARPP-21A and ARPP-21B. The amino acid sequence of purified bovine ARPP-21B has now been determined by gas-phase sequencing. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage with trypsin, chymotrypsin, subtilisin, and endoproteinase Lys-C. The resulting peptides were purified by high-performance liquid chromatography, and selected peptides were subjected to amino acid analysis and/or amino acid sequencing by automated Edman degradation. ARPP-21B consists of a single NH2-terminal blocked polypeptide chain of 88 residues, with a calculated molecular mass of 9561 Da, including an NH2-terminal acetyl group inferred by deblocking with an acylaminopeptidase. This molecular mass is significantly lower than earlier estimates based on SDS-PAGE or hydrodynamic measurements. The seryl residue phosphorylated by cAMP-dependent protein kinase (Hemmings et al., 1989) is located at position 55. The molecule contains 1 cysteinyl residue, at position 71, and contains no methionyl, tyrosyl, phenylalanyl, tryptophanyl, or histidinyl residues. Determination of the primary structure of ARPP-21, one of several phosphoproteins localized to dopaminoceptive neurons in the basal ganglia, provides a framework for further investigations into the molecular mechanisms involved in dopaminergic neurotransmission.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Brain / metabolism*
  • Cattle
  • Caudate Nucleus / metabolism*
  • Chromatography, High Pressure Liquid
  • Chymotrypsin / pharmacology
  • Cyclic AMP / physiology*
  • Dopamine / physiology*
  • Endopeptidases / pharmacology
  • Metalloendopeptidases*
  • Molecular Sequence Data
  • Phosphoproteins / analysis
  • Phosphoproteins / genetics*
  • Phosphoproteins / metabolism*
  • Subtilisins / pharmacology
  • Trypsin / pharmacology

Substances

  • Phosphoproteins
  • cyclic AMP-regulated phosphoprotein ARPP-21
  • Cyclic AMP
  • Endopeptidases
  • Subtilisins
  • Chymotrypsin
  • Trypsin
  • Metalloendopeptidases
  • peptidyl-Lys metalloendopeptidase
  • Dopamine