Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5220209

Senogles, S E; Caron, M G

The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..S binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.

• Research Organization: Duke Univ. Medical Center, Durham, NC
• OSTI ID: 5220209
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALKALOIDS
AMINES
ANALGESICS
ANIMALS
ANTIGENS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZAARENES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARDIOTONICS
CARDIOVASCULAR AGENTS
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM DEPRESSANTS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
DOPAMINE
DRUGS
ESTERS
EVEN-ODD NUCLEI
GUANINE
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPIDS
MAMMALS
MATERIALS
MEMBRANE PROTEINS
MEMBRANES
MORPHINE
NARCOTICS
NEUROREGULATORS
NUCLEI
NUCLEOTIDES
OPIUM
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PHENOLS
PHOSPHOLIPIDS
POLYPHENOLS
PROTEINS
PURIFICATION
PURINES
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RUMINANTS
SEPARATION PROCESSES
STOICHIOMETRY
SULFUR 35
SULFUR ISOTOPES
SYMPATHOMIMETICS
TOXIC MATERIALS
TOXINS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES