Cis- and Trans-Activation of Hormone Receptors: the LH Receptor

103

and the ʈIstituto di Neurobiologia Consiglio Nazionale delle Ricerche, Rome 00137, Italy Homo-and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product ␤) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein-and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers.Homo-or heterodimerization of G protein-coupled receptors (GPCRs) 1 has recently emerged as a constitutive or ligandinduced property of several receptor types. Receptor oligomerization has functional implications in terms of cell surface expression, ligand binding, signaling, and receptor trafficking (1). Many different GPCRs have been proved to undergo homoor heterodimer formation; these include the receptors for dopamine which can form both homo-(2-4) and heterodimers with somatostatin receptors (5), angiotensin A1 and A2 (6), adrenergic (7,8), GABA B (9 -11), ␦-and -opioid (12-15), rhodopsin (16), mGlu (17), vasopressin V2 (18), vasopressin/ossitocin (19), muscarinic (20), glucagon (21), Ca 2ϩ (22) sphingosine 1-phosphate (23), and the chemokine CXCR4, CCR2, and CCR5 receptors (24 -28). The molecular mechanism of receptor oligomerization varies among different receptors and involves transmembrane (TM) domains as well as extramembrane regions. Cysteine residues in the fourth TM segment are responsible for D2 dopamine receptor dimerization (4), and disulfide bonds are also involved in the oligomerization of Ca 2ϩ (22), -opioid (13), sphingosine 1-phosphate (23), and V2 vasopressin receptors (18); sequen...

Section: Discussioncontrasting

confidence: 99%

Ligand-independent CXCR2 Dimerization

Trettel

Bartolomeo

Lauro

et al. 2003

103

“…Similarly, nonfunctional hormone-receptor complexes formed by binding of hCG to nonsignaling LH receptor mutants exhibit faster rotational diffusion (54) than do functional hormone-occupied LH receptors (55), no increase in FRET in response to ligand (4) and are not confined in either raft domains or membrane microdomains examined using single particle tracking (7). These results suggest that the slower lateral dynamics observed for Au-DG-hCG when cells are treated with hCG may result from a bystander effect on Au-DG-tagged receptors that may include direct interactions between hCG-occupied LH receptors and the Au-DG-hCG occupied receptors similar to LH receptor trans-activation described by Ji and co-workers (56).…”

Section: Discussionsupporting

confidence: 61%

Restricted Lateral Diffusion of Luteinizing Hormone Receptors in Membrane Microdomains

Wolf‐Ringwall

Winter

Liu

et al. 2011

Signal transduction by luteinizing hormone (LH)3 receptor plays an essential role in the normal reproductive function of both male and female mammals by promoting ovulation, follicle maturation, corpus luteum formation, and steroidogenesis. LH receptor signaling in response to saturating concentrations of LH or human chorionic gonadotropin (hCG) involves substantial changes in receptor lateral and rotational dynamics (1, 2) as well as the association of LH receptors with membrane rafts (3). FRET techniques (4) and electron microscopy (5) indicate that functional hormone-receptor complexes can also become self-associated into dimers/oligomers following ligand binding. Previous experimental strategies have examined changes in receptor motions on collections of cells or large numbers of fluorescently tagged molecules on single cells. It is now possible, however, to examine the lateral motions of individual LH receptors on living cells using microscope-based single particle tracking techniques (6).Although the mechanism involved in retention of hCG-occupied LH receptors in small membrane compartments is still unclear, exposure to saturating concentrations of hCG results in confinement of the majority of LH receptors within small cell surface compartments. LH receptors remain within these compartments for comparatively long times and appear to diffuse pseudo-randomly before being captured within another compartment of similar size (7). Similar behavior has been described and analyzed by Kusumi and co-workers (6) for selected phospholipids and for transferrin receptors (8) and by Daumas and co-workers (9) for the -opioid receptor. Daumas argues that the -opioid receptor can both diffuse within the bulk membrane and exhibit confinement within a microdomain that itself diffuses slowly. Kusumi and co-workers (6) suggest that particles may be confined by proteins forming a barrier that is either continuous or discontinuous, leading to receptor diffusion within small membrane regions accompanied by intermittent escape from compartments and periods of unrestricted diffusion in the bulk membrane.Our previous studies of LH receptor lateral and rotational diffusion suggest that actin microfilaments or membrane protein interactions with the cytoskeleton may provide organizing structures that restrict the lateral motions of receptors (1, 2). However, the mechanism of hormone-initiated LH receptor confinement in small compartments has not been examined previously in detail at the single molecule level. In the present work, we compared the lateral dynamics of individual LH receptors that were either FLAG-tagged and identified using FLAG-specific antibodies with the diffusion of native receptors occupied by gold nanoparticle-hCG conjugates (Au-hCG) or gold-deglycosylated hCG (Au-DG-hCG). The latter material is

“…Evidence pointing toward this goes back to as early as the 1980s when data from equilibrium sedimentation of detergent-solubilized LHR and radiation inactivation of LHRs on gonadal cells suggested that the LHR may be present in a dimeric or oligomeric form (31,32). More recently, functional complementation studies further suggested the dimerization of the LHR (33,34). Direct evidence in support of dimerization of the LHR has been obtained using fluorescent resonance energy transfer (FRET) (35)(36)(37)(38) as well as our studies demonstrating specific co-immunoprecipitation of differentially tagged forms of the LHR (39).…”

mentioning

confidence: 64%

Bioluminescence Resonance Energy Transfer Studies Reveal Constitutive Dimerization of the Human Lutropin Receptor and a Lack of Correlation between Receptor Activation and the Propensity for Dimerization

Guan

et al. 2009

Previous studies from our laboratory using co-immunoprecipitation techniques suggested that the human lutropin receptor (hLHR) constitutively self-associates into dimers/oligomers and that agonist treatment of cells either increased hLHR dimerization/oligomerization and/or stabilized hLHR dimers/ oligomers to detergent solubilization (Tao, Y. X., Johnson, N. B., and Segaloff, D. L. (2004) J. Biol. Chem. 279, 5904 -5914). In this study, bioluminescence resonance energy transfer (BRET 2 ) analyses confirmed that the hLHR constitutively self-associates in living cells. After subcellular fractionation, hLHR dimers/oligomers were detected in both the plasma membrane and endoplasmic reticulum (ER). Further evidence supporting the constitutive formation of hLHR dimer/oligomers in the ER is provided by data showing homodimerization of misfolded hLHR mutants that are retained in the ER. These mutants, when co-expressed with wild-type receptor, are shown by BRET 2 to heterodimerize, accounting for their dominant-negative effects on cell surface receptor expression. Hormone desorption assays using intact cells demonstrate allosterism between hLHR protomers, indicating functional cell surface hLHR dimers. However, quantitative BRET 2 analyses in intact cells indicate a lack of effect of agonist on the propensity of the hLHR to dimerize. Using purified plasma membranes, human chorionic gonadotropin was similarly observed to have no effect on the BRET 2 signal. An examination of the propensity for constitutively active and signaling inactive hLHR mutants to dimerize further showed no correlation between dimerization and the activation state of the hLHR. Taken altogether, our data suggest that hLHR dimers/ oligomers are formed early in the biosynthetic pathway in the ER, are constitutively expressed on the plasma membrane, and are not affected by the activation state of the hLHR.