The interaction of follicle stimulating hormone with its specific GPCR, the follicle stimulating hormone receptor (FSHR) is facilitated by the extracellular loops (ELs) which contact the transmembrane domain and relay the signal downstream. In order to determine the contribution of non conserved residues from the EL3 of FSHR in conferring specificity to FSH-FSHR interaction, they were swapped with respective residues from luteinizing hormone/choriogonadotropin receptor. The triple mutant EL3M exhibited increased internalization of FSH-FSHR complexes without affecting the cAMP signaling response. Here, substitution point mutants S588T, K589N and A590S of the EL3 of FSHR were generated and characterized. None of these substitutions affected FSHR expression, FSH binding ability and cAMP production as compared to wild type FSHR. However, the high internalization of EL3M was observed to be due to the K589N and A590S substitutions. Further, all the mutants showed an impaired FSH mediated ERK phosphorylation response and the extent of impairment was most striking in case of the A590S substitution. Interestingly, S588T mutant exhibited impaired ERK phosphorylation, without change in receptor internalization, indicating that these processes can be dissociated. Thus, the FSHR specific residues K589 and A590 in the EL3 of FSHR seem to be crucial for FSH-induced internalization and ERK phosphorylation.
Keywords: ERK phosphorylation; Extracellular loop 3; FSH receptor; Internalization.
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