Follice-stimulating hormone receptor forms oligomers and shows evidence of carboxyl-terminal proteolytic processing.
FSH receptor (FSHR), a member of the G protein-coupled receptor superfamily, is present in the plasma membrane of ovarian granulosa cells and testicular Sertoli cells. FSH regulates normal ovarian follicle development and spermatogenesis through FSHR. The extracellular domain of FSHR is a weakly associated homodimer in the recently solved crystal structure of FSH in complex with the extracellular domain of FSHR. However, there is currently no biochemical data that demonstrate that FSHR exists as a dimer or higher-order oligomer in cell membranes. A fluorescence resonance energy transfer assay was used to determine whether full-length native FSHR is an oligomer. FSHR-specific monoclonal antibody or Fab fragments, labeled with two different fluorophores, allowed the study of nontagged receptor in situ. Unoccupied FSHR exhibited strong fluorescence resonance energy transfer profiles in situ. Complementary coimmunoprecipitation experiments of myc- or FLAG-tagged FSHR indicated that FSHR forms oligomers early in receptor biosynthesis. No effect of FSH treatment was observed. Thus, immature forms of FSHR, not yet fully processed, were observed to coimmunoprecipitate. An unexpected observation was made that the C-terminal epitope tags are removed from FSHR before arrival at the cell surface. These results provide the first evidence for oligomers of full-length FSHR in situ and for C-terminal proteolytic processing of FSHR and that both events take place during biosynthesis. This may explain how heterozygous mutations in the FSHR gene that affect receptor trafficking may be ameliorated by oligomer formation.
Thomas
Richard M
RM
Wadsworth Center, David Axelrod Institute for Public Health, New York State Department of Health, Albany, New York 12208, USA.
Nechamen
Cheryl A
CA
Mazurkiewicz
Joseph E
JE
Muda
Marco
M
Palmer
Stephen
S
Dias
James A
JA
D019921:Biotinylation; D002460:Cell Line; D019281:Dimerization; D000939:Epitopes; D031541:Fluorescence Resonance Energy Transfer; D006579:Heterozygote; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011962:Receptors, FSH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000939:Epitopes; D011956:Receptors, Cell Surface; D011962:Receptors, FSH