title: Decreased degradation of internalized follicle-stimulating hormone caused by mutation of aspartic acid 6.30(550) in a protein kinase-CK2 consensus sequence in the third intracellular loop of human follicle-stimulating hormone receptor.
abstract: A naturally occurring mutation in follicle-stimulating hormone receptor (FSHR) gene has been reported: an amino acid change to glycine occurs at a conserved aspartic acid 550 (D550, D567, D6.30(567)). This residue is contained in a protein kinase-CK2 consensus site present in human FSHR (hFSHR) intracellular loop 3 (iL3). Because CK2 has been reported to play a role in trafficking of some receptors, the potential roles for CK2 and D550 in FSHR function were evaluated by generating a D550A mutation in the hFSHR. The hFSHR-D550A binds hormone similarly to WT-hFSHR when expressed in HEK293T cells. Western blot analyses showed lower levels of mature hFSHR-D550A. Maximal cAMP production of both hFSHR-D550A as well as the naturally occurring mutation hFSHR-D550G was diminished, but constitutive activity was not observed. Unexpectedly, when (125)I-hFSH bound to hFSHR-D550A or hFSHR-D550G, intracellular accumulation of radiolabeled FSH was observed. Both sucrose and dominant-negative dynamin blocked internalization of radiolabeled FSH and its commensurate intracellular accumulation. Accumulation of radiolabeled FSH in cells transfected with hFSHR-D550A is due to a defect in degradation of hFSH as measured in pulse chase studies, and confocal microscopy imaging revealed that FSH accumulated in large intracellular structures. CK2 kinase activity is not required for proper degradation of internalized FSH because inhibition of CK2 kinase activity in cells expressing hFSHR did not uncouple degradation of internalized radiolabeled FSH. Additionally, the CK2 consensus site in FSHR iL3 is not required for binding because CK2alpha coimmunoprecipitated with hFSHR-D550A. Thus, mutation of D550 uncouples the link between internalization and degradation of hFSH.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, USA.
authors.forename: Kerri S
authors.initials: KS
authors.lastname: Kluetzman
authors.forename: Richard M
authors.initials: RM
authors.lastname: Thomas
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Nechamen
authors.forename: James A
authors.initials: JA
authors.lastname: Dias
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry*; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D011962:Receptors, FSH / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011962:Receptors, FSH; D001224:Aspartic Acid; D005640:Follicle Stimulating Hormone; D047390:Casein Kinase II