Intrinsically Fluorescent Luteinizing Hormone Receptor Demonstrates Hormone-Driven Aggregation

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20°C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 ؎ 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 ؎ 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with ␤-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30 -35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.The general mechanisms underlying the activation of Gprotein-coupled receptors (GPCRs) 1 and the following attenuation of the cellular response seem well established at the molecular level. The prevailing view is that the intracellular signal results from a cascade of transient and sequential molecular interactions starting with the activation of the heterotrimeric G-protein upon ligand/receptor interaction. The biological effect then can be attenuated by three main mechanisms: (i) agonists removal; (ii) agonist-mediated desensitization of the receptor molecules by uncoupling of the activated receptors from heterotrimeric G-proteins; and (iii) agonist-stimulated receptor endocytosis followed by either recycling or degradation. The remaining key questions are to understand the architecture governing the molecular interactions of the signaling cascade and how these multiple interactions take place in space and time, notably starting with the lateral organization of the partners at the level of the plasma membrane (1-4). Here we investigate these different aspects using the neurokinin NK2 receptor expressed in HEK293 cells as a model system.Neurokinins form a family of neuropeptides acting at three distinct GPCRs, namely NK1, NK2 and NK3. The NK2 receptor shows preferential binding for the endogenous neurokinin A (NKA) (5) and is mostly found in peripheral nervous tissues where it participates mainly to smooth muscle contraction. In pr...

Section: Resultssupporting

confidence: 88%

Dynamic Confinement of NK2 Receptors in the Plasma Membrane

Cézanne

Lecat

Lagane

et al. 2004

“…This effect is in total opposition with that of DAMGO, which increases MOP receptor compartmentalization in SH-SY5Y cells (40) 4 and with that of agonists in general. Stimulation by agonist slows down the diffusion of chemokine CXCR1 (57) and luteinizing hormone receptors (58), even leading to immobilization in the case of neurokinin NK2 receptors (26), and/or accentuates receptor confinement by increasing the immobile fraction (57,58) or by reducing the size of the diffusion domain (26). In contrast, the effect of 1DMe rather looks like the action of an antagonist, which produces no effect in the case of serotonin receptors (59) or increases the mobile fraction in the case of N-formyl peptide receptors (60), suggesting therefore that the functional blockade and the mobility change of opioid receptors by NPFF could be linked.…”

Section: Discussionmentioning

confidence: 99%

Physical Association between Neuropeptide FF and μ-Opioid Receptors as a Possible Molecular Basis for Anti-opioid Activity

Roumy

Lorenzo

Mazères

et al. 2007

Neuropeptide FF (NPFF) modulates the opioid system by exerting functional anti-opioid activity on neurons, the mechanism of which is unknown. By using a model of SH-SY5Y cells, we recently postulated that anti-opioid activity likely takes place upstream from the signaling cascade, suggesting that NPFF receptors could block opioid receptors by physical interaction. In the present study, fluorescence techniques were used to monitor the physical association and the dynamic of NPFF 2 and -opioid (

“…Based on our observation that exposure of follicular membranes to 8% ethanol accelerates the rate and increases the extent of LHR desensitization (23), coupled with evidence that the receptor appears to aggregate under conditions that most likely lead to receptor desensitization (26,29,31), we hypothesized that conversion of the receptor from an active to a desensitized state was associated with oligomerization of the LHR into higher ordered structures. We used a plasma membrane model in which endogenous receptors are localized at the plasma membrane.…”

Section: Discussionmentioning

confidence: 99%

Membrane Organization of Luteinizing Hormone Receptors Differs between Actively Signaling and Desensitized Receptors

Hunzicker-Dunn

Barisas

Song

et al. 2003

Self Cite

Signaling by the luteinizing hormone/choriogonadotropin receptor (LHR) is of considerable interest because of its requirement for successful reproduction. Time-resolved phosphorescence anisotropy and fluorescence resonance energy transfer were used to investigate the organization of endogenous LHRs in porcine follicular membranes in two distinct signaling states, active and desensitized. Desensitized LHRs exhibited ϳ3-fold slower rotational correlation times compared with active LHRs (59 ؎ 4 and 21 ؎ 9 s, respectively), suggesting that with agonist-dependent desensitization the receptors are organized into larger protein complexes. Incubation of membranes with inhibitors of LHR desensitization, such as neutralizing anti-arrestin antibodies, a synthetic peptide corresponding to the third intracellular loop of the LHR but not the corresponding scrambled peptide, or catalytically inactive ARNO, resulted in faster rotational diffusion times equivalent to those of actively signaling LHRs. Furthermore, desensitized LHRs exhibited a 2.4-fold increase in fluorescence resonance energy transfer between LHRs suggesting that the larger protein aggregates formed during desensitization contain more self-associated LHRs. These results indicate that agonist-dependent LHR desensitization precedes organization of LHRs at the cells surface into larger protein aggregates. The luteinizing hormone/choriogonadotropin receptor (LHR)1 is a seven transmembrane-spanning receptor coupled to G-proteins, most typically the stimulatory guanine nucleotide-binding proteins, which activate adenylyl cyclase to produce the second messenger signal, cAMP (1). Mechanisms involved in signal transduction by the LHR are of considerable interest because of the importance of the receptors in mammalian reproduction. In females, activation of the LHR regulates terminal follicular development and promotes ovulation and during pregnancy maintains progesterone production by the corpus luteum (2). In males, LHR signaling promotes testosterone production by Leydig cells (2). Signal transduction by the LHR to G s can be tempered or desensitized as a consequence of arrestin2 (␤-arrestin1) binding to the receptor at the third intracellular (3i) loop (3). LHR desensitization occurs in ovarian follicles in response to the mid-cycle LH surge that promotes ovulation (4) and, based on serum progesterone levels, appears to occur in human corpora lutea in response to elevated levels of hCG during pregnancy (5).The ␤ 2 -adrenergic receptor has served as a model for desensitization of G-protein-coupled receptors (GPCRs). Following agonist activation, desensitization of the ␤ 2 -adrenergic receptor is triggered by receptor phosphorylation catalyzed by a G-protein receptor kinase (6, 7). The G-protein receptor kinase phosphorylation sites on the ␤ 2 -adrenergic receptor facilitate interaction of the receptor with non-visual arrestins (8). Arrestins sterically hinder the ability of the receptor to activate G-proteins and thus moderate signal transduction by GPCRs (9, 10). Howe...