Evidence from photoaffinity labelling studies for coupling of the alpha/sub 1/-adrenergic receptor to a guanine-nucleotide (G) binding protein

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

OSTI ID:5151411

Graham, R M; Sena, L; Schwarz, K R; Homcy, C J

In contrast to ..beta..- and ..cap alpha../sub 2/-adrenergic receptors the role of a G-protein in signal transduction at ..cap alpha..-adrenergic receptors has been difficult to define. Using rat hepatic membranes prepared to avoid retention of endogenous nucleotides and activation of Ca/sup 2 +/-sensitive proteases, a Gpp(NH)p shift in agonist ((-)epinephrine) affinity from an IC/sub 50/ 10/sup -6/ to 5 x 10/sup -5/ M was readily demonstrable in competition studies with the ..cap alpha../sub 1/-specific radioligand (/sup 3/H)prazosin, but was not observed in membranes prepared without protease inhibitors (PIs). Labelling of these membranes with the photolabile prazosin analog, (/sup 125/I)CP65,526, followed by SDS-PAGE/autoradiography revealed a predominant, specifically labelled protein of M/sub r/ = 80,000, whereas a M/sub r/ = 59,000 peptide was evident with membranes prepared in the absence of PIs. The IC/sub 50/ for inhibition of labelling of the M/sub r/ = 80,000 peptide by (-)epinephrine, as determined by radiochromatogram scanning of autoradiographs of the photolabelled receptor, shifted from 10/sup -7/ to 10/sup -6/ in the presence of Gpp(NH)p. However, no shift in agonist affinity at the M/sub r/ = 59,000 peptide was evident in membranes prepared without PIs. This approach provides visual evidence for a G-protein-mediated shift in agonist affinity at the ..cap alpha../sub 1/-adrenergic receptor and allows a correlation between subunit size analysis and ligand binding.

• Research Organization: Massachusetts General Hospital, Boston
• OSTI ID: 5151411
• 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
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL SHIFT
CPB
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ENZYME INHIBITORS
GLANDS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LABELLING
LIGANDS
LIVER
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PROTEINS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SYMPATHOMIMETICS
TRITIUM COMPOUNDS
VERTEBRATES