Restricted Lateral Diffusion of Luteinizing Hormone Receptors in Membrane Microdomains

Amber L Wolf-Ringwall,Peter W Winter,Jingjing Liu,Alan K Van Orden,Deborah A Roess,B George Barisas

doi:10.1074/jbc.m111.250969

Amber L Wolf-Ringwall, Peter W Winter + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m111.250969

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Abstract

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.

Highlights

Cle maturation, corpus luteum formation, and steroidogenesis
Individual FLAG-luteinizing hormone (LH) receptors on cells treated with 100 nM human chorionic gonadotropin (hCG) exhibited significantly slower lateral diffusion (Fig. 1, lower panel)
We have previously shown that binding of saturating concentrations of hCG to rat LH-WT receptors results in redistribution of essentially all LH receptors from the bulk membrane into cholesterol-enriched membrane rafts [7] and that LH or hCG treatment causes a marked reduction in the fraction of mobile receptors [44]

Summary

EXPERIMENTAL PROCEDURES

Materials and Cell Culture—CHO cells were maintained in high glucose DMEM (Mediatech, Inc., Manassas, VA) supplemented with 10% FBS, 2 mM L-glutamine, 100 units of penicillin/ml, 100 ␮g streptomycin/ml (Gemini Bio-Products, Woodland, CA), and 1ϫ minimum Eagle’s medium nonessential amino acid solution (Sigma-Aldrich). For homo-FRET, FCS, and PCH experiments, we used a stable CHO cell line expressing human YFP-LHR at the C terminus as described previously [16]. Fluorescence Correlation Spectroscopy and Photon-counting Histogram Analysis—PCH analysis was used to evaluate oligomerization of YFP-LHR stably expressed on CHO cells in response to increasing concentrations of hormone. CHO cells stably expressing YFP-LHR were grown to 50% confluence on sterile dishes and examined directly Both FCS and PCH experiments were performed on a modified Nikon TE1000 inverted microscope equipped with a 100ϫ, 1.25 numerical aperture, oil immersion objective, an Omnichrome MellesGriot multiline air-cooled argon ion laser operating at 514.5 nm, two 570/32 nm band pass filters, two PerkinElmer Life. Weighted least-squares fitting was used to obtain estimates of Neff, the average molecular brightness (⑀) of detected particles and the out-of-focus emission ratio (F) as described previously (29 –31)

RESULTS

24 Ϯ 21 24 Ϯ 20 21 Ϯ 17 20 Ϯ 13 27 Ϯ 26 25 Ϯ 18

25 Ϯ 18 28 Ϯ 20 27 Ϯ 21 22 Ϯ 16

25 Ϯ 18 29 Ϯ 14 29 Ϯ 12 32 Ϯ 1

DISCUSSION