The D 2
-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein
Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca 2+ mobilization/phosphoinositide pathways. The D 2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) ([ 35S]GTPγS) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [ 32P]ADP-ribosylation of affinity-purified D 2 receptor preparations by pertussis toxin revealed the presence of a substrate of M(r) 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [ 32P]ADP-ribosylated endogenous N protein, transducin, and N(i) and N(o) from brain revealed similarities but not identity between the endogenous pituitary N protein and brain N(i) and N(o). Immunoblotting of the partially purified D 2 receptor preparations showed an M(r) 39,000-40,000 band with an N(i)-specific antiserum raised against a synthetic peptide, and with RV3, an N(o)-specific antiserum, but not with CW6, an antiserum strongly reactive with brain N(i). Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D 2 receptor. As measured by [ 35S]GTPγS binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D 2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that N(o) and/or a form of N(i) distinct from the M(r) 41,000 pertussis toxin substrate (N(i)) is the predominant N protein functionally coupled with the D 2-dopamine receptor of anterior pituitary.
Senogles, SE; Benovic, JL; Amlaiky, N; Unson, C; Milligan, G; Vinitsky, R; Spiegel, AM; Caron, MG
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