Critical involvement of the hinge region of the follicle-stimulating hormone receptor in the activation of the receptor.
The follicle-stimulating hormone receptor (FSHR) is a G-protein-coupled receptor with a large hormone-specific extracellular domain (amino acids (aa) 1-366) and a characteristic seven-transmembrane domain (TMD; aa 367-695). The extracellular domain is composed of leucine-rich repeats (LRRs; aa 18-259) connected to TMD by a hinge region (HinR; aa 260-366), whose role in the hormone action is not clearly understood. We generated a novel polyclonal HinR antibody that specifically stimulates cAMP production by HEK 293 cells expressing FSHR in a hormone-independent manner. The monovalent antibody retained the stimulatory potential. The segment of aa 296-331 in HinR was identified as the binding site for the stimulatory antibody. Deletions of this entire segment or any 10 amino acids within this segment from FSHR led to complete loss of antibody response and, surprisingly, response to the hormone as well despite all mutants exhibiting cell surface receptor density and affinity comparable with the wild type receptor. Interestingly, these mutants exhibited higher basal cAMP production. The mutant lacking LRRs with the intact HinR (Delta18-259) showed suppressed basal activity, which increased significantly with deletions extending to aa 331. These data suggest that the segment 296-331 acts as a tethered inverse agonist of the TMD and plays a very critical role in the hormonal activation of FSHR. The mutants lacking LRRs failed to bind FSH, whereas deletions in HinR had no effect on hormone binding, indicating that the LRRs constitute the primary high affinity binding site, whereas HinR may not play a significant role in FSH binding.
Agrawal
Gaurav
G
Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Dighe
Rajan R
RR
Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India. Electronic address: rdighe@mrdg.iisc.ernet.in.
D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D011962:Receptors, FSH / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011962:Receptors, FSH