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Self-Association and Raft Localization of Functional Luteinizing Hormone Receptors

Department of Biomedical Sciences and Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado 80523

	ABSTRACT

TOP ABSTRACT INTRODUCTION FUNCTIONAL LH RECEPTORS UNDERGO... DESENSITIZED LH RECEPTORS ARE... FUNCTIONAL LH RECEPTORS... DISCUSSION REFERENCES

 
Membrane motions of LH receptors following binding of hormone agonists are consistent with hormone-driven aggregation. It is increasingly apparent that G protein-coupled receptors, including the LH receptor, are engaged in dynamic interactions with one another and other membrane components. These interactions are governed, in part, by a number of factors including whether the receptor has bound ligand, whether the receptor is capable of transducing a hormone-mediated signal, and the nature of the membrane environment within which the receptor is found. Microscopic methods, including laser-optical techniques, are ideally suited to probe dynamic events on cell membranes and provide an opportunity to examine interactions between receptors and other membrane components on viable cells. We and others have used a variety of techniques, some of which are summarized below, to examine functional and nonfunctional LH receptors on viable cells and the membrane environment of these receptors during cell signaling events.

luteinizing hormone, mechanisms of hormone action, signal transduction

	INTRODUCTION

TOP ABSTRACT INTRODUCTION FUNCTIONAL LH RECEPTORS UNDERGO... DESENSITIZED LH RECEPTORS ARE... FUNCTIONAL LH RECEPTORS... DISCUSSION REFERENCES

 
The ability of an LH receptor to bind ligand and transduce signal directly affects the types of interactions evoked by the receptor. LH receptor function can be substantively altered by several strategies including deleting portions of the receptor, substituting amino acids within the receptor, or binding a nonfunctional ligand such as deglycosylated hCG to the receptor. Functional LH receptors bind hormone agonists and produce measurable increases in levels of downstream signaling molecules such as cAMP. Nonfunctional hormone receptor complexes include receptors that have bound hormone antagonists or receptors that remain uncoupled from signal transduction machinery despite binding of hormone agonists. To determine whether the membrane environment of nonfunctional hormone receptor complexes differed qualitatively from that of functional hormone receptor complexes, we compared the rotational dynamics of various complexes on 293 cells using time-resolved phosphorescence anisotropy methods. Because the rotational diffusion of membrane proteins is linearly related to the in-membrane volume of the complex identified by the phosphorescent probe [1], rotational diffusion measurements are sensitive indicators of protein-protein associations that might be critical for receptor function. Binding of hCG to the rat wild-type receptor expressed on 293 cells (LHR-wt cells) produced functional receptors that exhibited rotational correlation times longer than 1000 µsec. However, nonfunctional hormone receptor complexes including wild-type LH receptors occupied by deglycosylated hCG, an hCG antagonist [2], or hCG bound to LH receptors containing a substitution of arginine for lysine at amino acid 583 (LHR-K583R) [3, 4] had significantly shorter rotational correlation times at 37°C (63 ± 8 µsec and 130 ± 12 µsec, respectively. These results suggest that functional LH receptors are present in membrane complexes that exhibit slow rotational diffusion or are rotationally immobile. Shorter rotational correlation times for nonfunctional hormone receptor complexes indicate smaller molecular weight complexes and may reflect the absence of essential interactions between these complexes and other membrane proteins.

	FUNCTIONAL LH RECEPTORS UNDERGO SELF-ASSOCIATION

TOP ABSTRACT INTRODUCTION FUNCTIONAL LH RECEPTORS UNDERGO... DESENSITIZED LH RECEPTORS ARE... FUNCTIONAL LH RECEPTORS... DISCUSSION REFERENCES

 
Although rotational methods are useful for qualitative assessment of LH receptor interactions with other membrane components, they do not provide direct information on the specific interactions occurring within, for example, slowly diffusing complexes. Because hormone-driven aggregation of G protein-coupled receptors like the LH receptor may be a necessary prelude to signal transduction, it is of interest to know, for example, whether functional LH receptors are also self-associated within slowly rotating membrane structures. To determine whether hormone-occupied LH receptors were self-associated into dimers or oligomers, we measured fluorescence-resonant energy transfer between fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TrITC)-derivatized LH or hCG using a method based on the reduced rate of irreversible photobleaching of FITC fluorophores when TrITC fluorophores are nearby [5]. Slower rates of fluorescence disappearance for cells labeled with the FITC fluorescence donor and TrITC fluorescence acceptor than for cells labeled with FITC only indicated energy transfer from fluorescence donor to acceptor, which occurs only when the donor and acceptor are separated by distances less than about 100 Å [6]. Binding of hormone to wild-type LH receptors on 293 cells was accompanied by self-aggregation of luteinizing hormone receptors as indicated by values for fluorescence resonant energy transfer efficiency of 13% to 17%. There was little or no energy transfer between LH-K583R receptors occupied by hCG, suggesting that productive signal transduction may require receptor-receptor interactions.

These studies support previous observations of LH receptor clustering in the plasma membrane following hormone binding. Elegant electron microscopy studies by Luborsky et al. [7] demonstrate that extensive association of LH receptors into structures containing multiple copies of the LH receptor occurs following exposure of rat luteal cells to high concentrations of LH. Similarly, immunofluorescence studies of the LH receptor on rat granulosa cells demonstrates the presence of large, punctate structures on the cell membrane following ligand binding [8, 9]. However, because fluorescent clusters containing LH receptors visible in light microscopy or ferritin clusters in electron microscopy [7] can represent structures containing multiple copies of the receptor in which the receptors are not in direct contact, fluorescence energy transfer measurements provide the first direct evidence for LH receptor association at distances of less than 100 Å.

These experiments raise the question of whether signal transduction by LH receptors requires a change in receptor organization from an isolated state to an aggregated state. To address this, we coupled the rat LH receptor to variants of green fluorescent protein (GFP) to produce an intrinsically fluorescent receptor. Using cell lines coexpressing receptors expressed with either GFP or yellow fluorescent protein (YFP), we have shown that there is fluorescence energy transfer between LH receptors if hormone binding to the LH receptor results in cAMP formation [10] and that there is no energy transfer between nonfunctional mutated LH receptors following binding of LH or hCG or between functional LH receptors occupied by deglycosylated hCG [11]. Thus, LH receptors that are competent to signal are in close proximity (less than 100 Å) to one another following binding of ligand. These results are consistent with studies showing dimerization of several G protein-coupled receptors including the ß2-adrenergic receptor [12], the -opioid receptor [13], and the M3 muscarinic receptor [14] as well as rescue of nonfunctional ß2-adrenergic receptors via dimerization [12].

	DESENSITIZED LH RECEPTORS ARE SELF-ASSOCIATED IN SLOWLY DIFFUSING COMPLEXES

TOP ABSTRACT INTRODUCTION FUNCTIONAL LH RECEPTORS UNDERGO... DESENSITIZED LH RECEPTORS ARE... FUNCTIONAL LH RECEPTORS... DISCUSSION REFERENCES

 
LH receptors, in response to binding of ligand, can also undergo time-dependent changes in their functional status and remaining within the plasma membrane. Like other G-protein coupled receptors including the ß-adrenergic receptor [15], the LH receptor becomes less responsive, i.e., desensitized, within minutes after binding of hCG or LH [16, 17]. The LH receptor exhibits desensitization in response to treatment with saturating hormone concentrations, a process that has been observed in vitro in granulosa, luteal, and Leydig cells [18–21]. In preovulatory follicles, the surge of LH, which induces ovulation [18, 22, 23], also desensitizes the LH receptor. Desensitization of the LH receptor is followed by a decrease in cellular cAMP, despite the presence of LH and is required for initiation of oocyte meiosis [24]. Once desensitized, hormone binding to LH receptors only minimally activates adenylate cyclase [18] despite the fact that adenylate cyclase is still functional and can be activated by other means [21]. Desensitization of LH receptors following brief exposure to hormone is initially characterized by uncoupling of the receptor from the signal transduction apparatus rather than by a decrease in receptor number. This uncoupling process can be induced in vitro. After hormone binding to receptor, washing cells with a low pH buffer removes hormone from the receptor. Although the receptors are functionally undamaged by this treatment and will subsequently bind hormone [20], desensitization has nonetheless occurred.

To determine whether the desensitized LH receptor was self-associated within large, slowly diffusing structures and whether those structures had to dissipate before the receptor could again respond to hormone, we examined both LH receptor lateral diffusion and receptor self-association during times, 1 to 4 h following brief exposure of the receptor to hormone, when the receptor was desensitized but before any measurable internalization of the receptor had occurred. Lateral motions of membrane proteins diffusing without constraint from the membrane are rapid with diffusion coefficients on the order of 10^-9cm² sec^-1. Such unconstrained protein also has high values for recovery of fluorescence after photobleaching of fluorescent probes. Diffusion of membrane proteins on the membranes of viable cells depends primarily and only logarithmically on the in-membrane volume of that protein. Thus, comparatively large changes in complex size are necessary to elicit a change in the rate of protein diffusion. Other protein features such as a large extracellular domain or the presence of a GFP tag on the C-terminus of the receptor add little or no drag on the protein's motions. However, interactions between a fluorescently tagged protein and other membrane proteins do significantly slow the rate of protein diffusion and can reduce the fraction of laterally mobile molecules. Thus, comparatively large changes in complex size are necessary to elicit a change in the rate of protein diffusion.

Prior to binding of ligand, rLH-GFP receptors had a diffusion coefficient of 1.6 ± 0.3 x 10^-9 cm² sec^-1 and a fluorescence recovery of 58% ± 4%, indicating that most receptors were laterally mobile (Fig. 1, upper panel). When cells expressing LH-GFP receptors were treated with 100 nM hCG for 30 min followed by removal of bound hormone by a low pH wash, there was no cAMP synthesis above basal levels in response to repeated hormone challenge for the next 4 h. For receptors desensitized by hCG treatment, fluorescence recovery decreased to less than 20% at 1 h, indicating that most receptors were laterally immobile. This response to hCG treatment was virtually identical to that of native ovine [25] and rat [26] LH receptors on luteal cells. Over the next 4 h, there were incremental increases in both the rate of diffusion and the fraction of mobile receptors for LH receptors desensitized by LH or hCG. After 5 h receptors were again responsive to hormone challenge as indicated by an increase in intracellular cAMP. At this time, values for diffusion coefficients and fluorescence recovery were indistinguishable from those of untreated receptors. Prior to ligand binding, there was no energy transfer between LH receptors, indicating that the receptors were not self-associated (Fig. 1, lower panel). However, upon receptor desensitization by hCG, energy transfer efficiency increased to 18% at 1 h. Energy transfer then decreased slowly over the next 4 h. By 5 h there was no significant energy transfer between receptors. Thus, brief hormone treatment resulted in LH receptor self-association and inclusion of receptors in slowly diffusing structures. After initial signaling, receptors were nonfunctional and clustered in complexes that persisted in the membrane for several hours. The receptor was hormone responsive only when it was no longer self-associated and had dissociated from slowly diffusing complexes.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Spot fluorescence photobleaching recovery measurements of rLHR-GFP lateral diffusion prior to hormone binding and following hCG-induced receptor desensitization. The upper panel shows the diffusion coefficient (D) for the GFP-LHR-wt over the time course of recovery from receptor desensitization and fluorescence recovery (% recovery) of GFP-LHR-wt at the corresponding times. Receptors desensitized by either ligand exhibited significantly slower D 1 h following desensitization. D increased over the next 4 h until D was indistinguishable from that of untreated LHR. Percent recovery decreased upon binding of hormone and then increased over time until 5 h by which time % recovery did not differ significantly from that of untreated receptor. The lower panel shows percent fluorescence resonant energy transfer efficiency (% FRET efficiency) for LH receptor fusion proteins before (0 h) and after initiation of receptor desensitization (1–5 h) by 10 nM hCG. At 0 and 5 h when receptors are hormone responsive, there is no significant energy transfer between LH receptors. When receptors were not hormone responsive, there was measurable energy transfer between LH receptors. This figure was adapted from previous published results [10]

	FUNCTIONAL LH RECEPTORS TRANSLOCATE INTO SPECIALIZED MEMBRANE MICRODOMAINS

TOP ABSTRACT INTRODUCTION FUNCTIONAL LH RECEPTORS UNDERGO... DESENSITIZED LH RECEPTORS ARE... FUNCTIONAL LH RECEPTORS... DISCUSSION REFERENCES

 
Lateral diffusion studies of GFP-LH-wt receptors suggested that most unoccupied LH receptors were laterally mobile with a fast rate of lateral diffusion and that binding of ligand slowed receptor lateral diffusion 6- to 10-fold and reduced the fraction of mobile receptors [27]. The dramatic slowing of receptor lateral diffusion and a reduction in the fraction of mobile receptors are consistent with receptor trapping in specialized membrane microdomains, i.e., rafts following ligand binding. Membrane rafts are high-buoyancy membrane fragments that float in sucrose gradients and are typically enriched with sphingomyelin and cholesterol [28] as well as glycosylphosphatidyl inositol (GPI)-anchored proteins [29]. Pralle et al. [30, 31] used optical tweezers to estimate the diameter of these domains at about 24 nm and showed that these domains have boundaries that can limit the lateral movement of specific membrane proteins on neurites. Molecules capable of binding cholesterol, including cyclodextrins, saponins, and filipin, disrupt these microdomains and cause a redistribution of GPI-anchored proteins within the plasma membrane [29]. On some cells, these domains contain high concentrations of the membrane proteins necessary for cell signaling such as G proteins [32] and adenylate cyclase [33]. Thus, it has been suggested that rafts may serve as signaling platforms in the plasma membrane. A number of plasma membrane receptors including the IgA receptor [34], epithelial growth factor receptor [35], and the tissue factor receptor [34] move into membrane rafts during signal transduction. Translocation of membrane proteins into rafts can be driven by receptor cross-linking in the absence of ligand. Fc receptors, on cross-linking, move into rafts containing tyrosine kinases and are phosphorylated [36].

To test whether the LH receptor partitions into membrane rafts following binding of ligand, we stably expressed a FLAG (Sigma-Aldrich, St. Louis, MO)-tagged rat LH receptor using a FLAG-rLH receptor vector kindly provided by Dr. K.J. Menon (University of Michigan, Ann Arbor). Stable expression of the FLAG-rLH-wt receptor made it possible to identify the LH receptor on Western blots in the unbound and bound state. The hCG-treated FLAG-rLH receptors were consistently located in 25% to 40% sucrose (Fig. 2) together with caveolin, a protein that serves as a lipid raft membrane marker. Unoccupied LH receptors were found in a high-density lipid membrane and were not colocalized with caveolin (data not shown). The appearance of LH receptors within rafts following binding of hormone is consistent with imaging of the wild-type LH receptor coupled to GFP. The unoccupied GFP-coupled LH receptor had very diffuse distribution in the plasma membrane and underwent striking redistribution into punctate fluorescent clusters on the membrane shortly after being exposed to hormone. Whether the preferential localization of plasma membrane proteins such as LH receptors into discrete domains in which molecular motions are restricted has functional significance in, for example, receptor desensitization, as is shown in our working model for LH receptor localization in the membrane (Fig. 3), remains an unanswered question.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Western blots of FLAG-rLH-wt receptor from Chinese hamster ovary (CHO) ells appearing in various sucrose density fractions after isopycnic centrifugation. The upper panel shows receptors from untreated CHO cells appearing in fractions 13 to 15 (approximately 50% to 65% sucrose). The lower panel shows receptors from hCG-treated CHO cells that consistently appeared in fractions 3 to 6 (12% to 25% sucrose) together with caveolin (data not shown).

View larger version (29K): [in this window] [in a new window]   FIG. 3. We developed a working model for LH receptor interactions following hormone binding as well as during desensitization and resensitization of the receptor based on the physical evidence described in this review. Resting LH receptors (A), located in the bulk plasma membrane rather than in membrane rafts, exhibit fast lateral diffusion and do not appear to be self-associated until they have bound hormone agonists (B). Binding of hormone also drives receptors into plasma membrane rafts. Whether the receptor translocates into these structures as receptor monomers or dimers is not known. Desensitization of the receptor in response to ß-arrestin binding (C) is associated with slow rotational diffusion of the receptor, presumably as a result of incorporation of LH receptors into large molecular weight complexes that may be located in rafts. Resensitization of the receptor (D) is characterized by a reduction in receptor-receptor interactions and fast rates of receptor lateral diffusion

	DISCUSSION

TOP ABSTRACT INTRODUCTION FUNCTIONAL LH RECEPTORS UNDERGO... DESENSITIZED LH RECEPTORS ARE... FUNCTIONAL LH RECEPTORS... DISCUSSION REFERENCES

 
These studies underscore the dynamic nature of the LH receptor's interactions within the plasma membrane, summarized in our working model shown in Figure 3, in response to binding of ligand, signaling, and subsequent receptor desensitization. They also raise a number of questions about the receptor and its interactions. Presumably, if rafts serve as signaling platforms, there are time-dependent interactions between the receptor and proteins that transduce hormone signal or desensitize the receptor to prevent further activation of adenylate cyclase. Whether these various proteins are available within rafts or move into or out of the raft environment in some orchestrated fashion is an intriguing question.

Self-association of the receptor is also a potentially interesting phenomenon from several points of view. On a molecular level, the association of LH receptors may require both appropriate receptor conformation as well as specific dimerization sequences on both receptors. There has been such a sequence proposed for the ß-adrenergic receptor [37], but this sequence is not contained within the transmembrane domains of the rat LH receptor and does not appear to be well conserved among G-protein coupled receptors generally [38]. Guo et al. [38] proposed that the fourth transmembrane domain of the dopamine D2 receptor may contain sites used in formation of D2 receptor homodimers.

Other questions of interest include the relationships between receptor aggregation and signaling and desensitization as well as membrane localization of receptors during receptor internalization. We show in Figure 3 that desensitized receptors, presumably following binding of ß-arrestin, are present in large molecular weight structures, which we hypothesize are localized in membrane rafts. This is based on a study in collaboration with Dr. Mary Hunzicker-Dunn at Northwestern University, which shows that LH receptors from porcine granulosa cells are present in large, slowly rotating complexes only when LH receptors are desensitized (M. Hunzicker-Dunn and D.A. Roess, unpublished results). Treatment of membranes with antibodies to ß-arrestin blocks LH receptor desensitization and results in receptor-containing complexes that are smaller and have fast rotational correlation times. A recent study of the ß-adrenergic receptor [39] has linked binding of ß-arrestin to desensitization of the receptor as well as to receptor internalization via clathrin-coated pits, thus raising the possibility that ß-arrestin may function as a critical molecule in receptor aggregation, raft localization, desensitization, and internalization. However, for LH receptors, these temporal events as well as the membrane sites involved in receptor binding to ligand, signaling via G proteins, receptor interaction with arrestins, and subsequent internalization are still being actively studied. Lastly, the time-dependent increase in lateral diffusion of the desensitized LH receptor and a decrease in the extent of receptor self-association are indicative of large complexes that slowly dissociate until free receptor is again available for interaction with ligand. Whether these complexes dissociate within specific membrane microdomains or move out of membrane rafts prior to dissociation is also not known. Biophysical methods, particularly those applicable to examining receptors on viable cells, will be valuable tools for resolving these questions.

	ACKNOWLEDGMENTS

 
We would like to acknowledge the contributions made by past and present members of Dr. Roess's research group: Ying Lei, Dr. Regina Horvat, Dr. Mary Cahill, Guy Hagen, and Dr. Cynthia J. Brady as well as Drs. Mary Hunzicker-Dunn and George Barisas, who have provided invaluable collaborative assistance. We would also like to thank the Endocrine Society for permission to use the data shown in Figure 1. These data were originally published elsewhere (Horvat RD, Barisas BG, Roess DA. Luteinizing receptors are self-associated in slowly diffusing complexes during receptor desensitization. Mol Endocrinol 2001; 15:534–542).

	FOOTNOTES

 
¹ Correspondence: Deborah A. Roess, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523. FAX: 970 491 1801; daroess{at}lamar.colostate.edu

Received: 30 April 2003.

First decision: 22 May 2003.

Accepted: 11 July 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION FUNCTIONAL LH RECEPTORS UNDERGO... DESENSITIZED LH RECEPTORS ARE... FUNCTIONAL LH RECEPTORS... DISCUSSION REFERENCES

 

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5. Young RM, Arnette JK, Roess DA, Barisas BG. Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics. Biophys J 1994 67:881-888[Abstract/Free Full Text]
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