ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Amino acid sequence of ARPP-21B from bovine caudate nucleus.
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.
Williams
K R
KR
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Hemmings
H C
HC
LoPresti
M B
MB
Greengard
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002421:Caudate Nucleus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D004298:Dopamine / Q000502:physiology*; D010450:Endopeptidases / Q000494:pharmacology; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D013381:Subtilisins / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D010750:Phosphoproteins; C061368:cyclic AMP-regulated phosphoprotein ARPP-21; D000242:Cyclic AMP; D010450:Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D004298:Dopamine