DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein: regional, tissue, and phylogenetic distribution

Abstract

Rabbit antisera and mouse monoclonal antibodies have been prepared to bovine DARPP-32 (dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein, Mr = 32,000), and used to study its regional, tissue, and phylogenetic distributions. The antibodies, none of which distinguished between dephospho-DARPP-32 and phospho-DARPP-32, were characterized and used to develop a sensitive and specific radioimmunoassay for DARPP-32. The radioimmunoassay, in conjunction with immunolabeling of SDS/PAGE transfers and immunoprecipitation of phosphorylated tissue extracts, was used to measure immunoreactive DARPP-32 in microdissected regions of rat CNS, in peripheral nervous and non-nervous tissues, and in CNS tissue from various animal species. The distribution of DARPP-32 was generally consistent with the interpretation that it is localized primarily to dopaminoceptive cells that possess dopamine-sensitive adenylate cyclase (D-1 dopamine receptors coupled to adenylate cyclase). Within the rat CNS, DARPP-32 was most highly concentrated in the basal ganglia. DARPP-32 was present in neostriatum from all six mammalian species tested (mouse, rat, guinea pig, rabbit, cow, and rhesus monkey) at concentrations of from 96 to 144 pmol/mg total protein, which constituted from 0.22 to 0.32% of the total protein. DARPP-32 was also identified at low levels in several peripheral tissues, including choroid plexus, parathyroid cells, adrenal chromaffin cells, posterior pituitary gland, pineal gland, and superior cervical sympathetic ganglion. A phylogenetic survey was carried out of proteins immunologically related to DARPP-32 in nervous tissue from nonmammalian species. DARPP-32-like proteins were identified in dopaminoceptive brain regions from representative members of the amniote vertebrate classes (birds and reptiles), while none was identified in dopaminoceptive brain regions from representative members of the anamniote vertebrate classes (bony fishes and amphibians) or in nervous tissue from representative members of several invertebrate classes.

Activation of the cAMP/PKA/DARPP-32 Signaling Pathway is Required for Morphine Psychomotor Stimulation but not for Morphine Reward

“…PhosphoThr34-DARPP-32 and phosphoThr75-DARPP-32 were detected using a monoclonal (Snyder et al, 1992) and a polyclonal (Bibb et al, 1999) antibody, respectively. A monoclonal antibody generated against DARPP-32, which is not phosphorylation state specific, was used to estimate the total amount of DARPP-32 (Hemmings and Greengard, 1986). Antibody binding was revealed using goat antimouse HRP-linked IgG (diluted 1:10 000) and the ECL immunoblotting detection system.…”

Cannabinoid Action Depends on Phosphorylation of Dopamine- and cAMP-Regulated Phosphoprotein of 32 kDa at the Protein Kinase A Site in Striatal Projection Neurons

“…PhosphoThr34-DARPP-32 and phosphoThr75-DARPP-32 were detected using a monoclonal (Snyder et al, 1992) and a polyclonal (Bibb et al, 1999) antibody, respectively. A monoclonal antibody generated against DARPP-32, which is not phosphorylation state specific, was used to estimate the total amount of DARPP-32 (Hemmings and Greengard, 1986). Antibody binding was revealed using goat anti-mouse HRP-linked IgG (diluted 1:10,000) and the ECL immunoblotting detection system.…”

Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

“…The membranes were immunoblotted using antibodies against phospho-Ser235/236-rpS6, phospho-Thr389-S6K, phosphoThr202/Tyr204-ERK1/2 (Cell Signaling Technology, Beverly, MA), and phospho-Thr34-DARPP-32 (Snyder et al, 1992). Antibodies against rpS6, S6K (Cell Signaling Technology), and DARPP-32 (Hemmings and Greengard, 1986) that are not phosphorylation state specific were used to estimate the total amount of proteins. Detection was based on fluorescent secondary antibody binding and quantified using a Li-Cor Odyssey infrared fluorescent detection system (Li-Cor, Lincoln, NE) or with enhanced chemiluminescence reagent (GE Healthcare).…”