Subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ
Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs.
- Research Organization:
- Albany Medical College of Union Univ., NY
- OSTI ID:
- 6292096
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 26
- Country of Publication:
- United States
- Language:
- English
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FSH
RECEPTORS
LABELLING
MOLECULAR STRUCTURE
ACYLATION
AFFINITY
AUTORADIOGRAPHY
AZIDES
CATTLE
CELL MEMBRANES
CENTRIFUGATION
COVALENCE
CROSS-LINKING
ELECTROPHORESIS
IODINE ISOTOPES
MOLECULAR WEIGHT
PHOTOLYSIS
TESTES
ANIMALS
BODY
CELL CONSTITUENTS
CHEMICAL REACTIONS
DECOMPOSITION
DOMESTIC ANIMALS
GONADOTROPINS
GONADS
HORMONES
ISOTOPES
MALE GENITALS
MAMMALS
MEMBRANES
NITROGEN COMPOUNDS
ORGANS
PEPTIDE HORMONES
PHOTOCHEMICAL REACTIONS
PITUITARY HORMONES
POLYMERIZATION
RUMINANTS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques