Evidence from photoaffinity labelling studies for coupling of the alpha/sub 1/-adrenergic receptor to a guanine-nucleotide (G) binding protein
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-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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CPB
ENZYME INHIBITORS
IODINE 125
LIGANDS
LIVER
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TRITIUM COMPOUNDS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BODY
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DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
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INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
KINETICS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
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REACTION KINETICS
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550201* - Biochemistry- Tracer Techniques