title: Single-molecule analyses of fully functional fluorescent protein-tagged follitropin receptor reveal homodimerization and specific heterodimerization with lutropin receptor.
abstract: We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.
authors.affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Mazurkiewicz
authors.affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York.
authors.forename: Katharine
authors.initials: K
authors.lastname: Herrick-Davis
authors.affiliation: Center for Cardiovascular Science, Albany Medical College, Albany, New York.
authors.forename: Margarida
authors.initials: M
authors.lastname: Barroso
authors.affiliation: Research Support Network, Instituto Nacional de Ciencias Médicas y Nutrición SZ-Universidad Nacional Autónoma de México, México D.F., México.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Ulloa-Aguirre
authors.affiliation: Division of Genetic Disorders, Wadsworth Center, New York State Department of Health, Albany, New York.
authors.forename: Barbara
authors.initials: B
authors.lastname: Lindau-Shepard
authors.affiliation: Department of Biomedical Sciences, State University of New York at Albany, Albany, New York.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Thomas
authors.affiliation: Department of Biomedical Sciences, State University of New York at Albany, Albany, New York jdias@albany.edu.
authors.forename: James A
authors.initials: JA
authors.lastname: Dias
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002678:Chimera / Q000235:genetics; D000242:Cyclic AMP / Q000096:biosynthesis; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism; D011962:Receptors, FSH / Q000737:chemistry / Q000378:metabolism*; D011974:Receptors, LH / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011956:Receptors, Cell Surface; D011962:Receptors, FSH; D011974:Receptors, LH; D049452:Green Fluorescent Proteins; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP
keywords: follicle-stimulating hormone (FSH/FSH receptor); gonadotropins; granulosa cells; mechanisms of hormone action; oocyte maturation