The mobile receptor hypothesis revisited: a mechanistic role for hormone receptor lateral mobility in signal transduction.

Recent application of the technique of fluorescence photobleaching recovery to direct measurement of the lateral mobility of plasma membrane-localized hormone receptors has shed new light on the role of receptor lateral mobility in signal transduction. Receptors for insulin and EGF have been known for some time to be largely immobile at physiological temperatures. This presumably relates to their signal transduction mechanism, which appears to require intermolecular autophosphorylation (receptor aggregation) for activation. In contrast, G-protein coupled receptors must interact with other membrane components to bring about signal transduction, and it is interesting in this regard that the adenylate cyclase (AC) activating vasopressin V2-receptor is highly laterally mobile at 37 degrees C. It has recently been possible to reversibly modulate the V2-receptor mobile fraction (f) to largely varying extents, and to demonstrate thereby a direct effect on the maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin. A direct correlation between f and maximal cAMP production indicates that f may be a key parameter in hormone signal transduction in vivo, especially at sub-KD (physiological) hormone concentrations, with mobile receptors being required to effect G-protein activation.     

A mechanistic role for polypeptide hormone receptor lateral mobility in signal transduction

Summary Lateral diffusion of membrane-integral receptors within the plane of the membrane has been postulated to be mechanistically important for signal transduction. Direct measurement of polypeptide hormone receptor lateral mobility using fluorescence photobleaching recovery techniques indicates that tyrosine kinase receptors are largely immobile at physiological temperatures. This is presumably due to their signal transduction mechanism which requires intermolecular autophosphorylation through receptor dimerization and thus immobilization for activation. In contrast, G-protein coupled receptors must interact with other membrane components to effect signal transduction, and consistent with this, the phospholipase C-activating vasopressin V₁- and adenylate cyclase activating V₂-receptors are highly laterally mobile at 37°C. Modulation of the V₂-receptor mobile fraction (f) has demonstrated a direct correlation between f and receptor-agonist-dependent maximal cAMP productionin vivo at 37°C. This indicates that f is a key parameter in hormone signal transduction especially at physiological hormone concentrations, consistent with mobile receptors being required to effect V₂-agonist-dependent activation of G-proteins. Measurements using a V₂-specific antagonist show that antagonist-occupied receptors are highly mobile at 37°C, indicating that receptor immobilization is not the basis of antagonism. In contrast to agonist-occupied receptor however, antagonistoccupied receptors are not immobilized prior to endocytosis and down-regulation. Receptors may thus be freely mobile in the absence of agonistic ligand; stimulation by hormone agonist results in receptor association with other proteins, probably including cytoskeletal components, and immobilization. Receptor immobilization may be one of the important steps of desensitization subsequent to agonistic stimulation, through terminating receptor lateral movement which is instrumental in generating and amplifying the initial stimulatory signal within the plane of the membrane.Abbreviations FBR fluorescence photobleaching recovery - EGF epidermal growth factor - AC adenylate cyclase - D apparent lateral diffusion coefficient - f mobile fraction - G- GTP-binding protein - Gs stimulatory G-protein - TKR tyrosine kinase receptor - PDGF platelet-derived growth factor - IL interleukin     

Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells

The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.     

The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.

The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and beta-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor.

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.     

The stimulatory Gα(s) protein is involved in olfactory signal transduction in Drosophila

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology, constituting a key difference between the olfactory systems of insects and other animals. While heteromeric insect ORs form ligand-activated non-selective cation channels in recombinant expression systems, the evidence for an involvement of cyclic nucleotides and G-proteins in odor reception is inconsistent. We addressed this question in vivo by analyzing the role of G-proteins in olfactory signaling using electrophysiological recordings. We found that Gα_s plays a crucial role for odorant induced signal transduction in OR83b expressing olfactory sensory neurons, but not in neurons expressing CO₂ responsive proteins GR21a/GR63a. Moreover, signaling of Drosophila ORs involved Gα_s also in a heterologous expression system. In agreement with these observations was the finding that elevated levels of cAMP result in increased firing rates, demonstrating the existence of a cAMP dependent excitatory signaling pathway in the sensory neurons. Together, we provide evidence that Gα_s plays a role in the OR mediated signaling cascade in Drosophila.     

Regulation of water permeability of collecting ducts in mouse kidney during postnatal development

Water permeability of epithelial cells and response to vasopressin was studied on isolated fragments of collecting ducts in the kidney of C57BL/6J mice of three age groups (9, 18, and 60–90 days). The coefficient of osmotic water permeability P _f was evaluated from the rate of cell swelling after medium osmolality was changed from 300 to 200 mOsm/l. The P _f value proved to be significantly lower in mice at the age of 9 days than at the age of 18 days, i.e., after the transition to mixed feeding; although P _f at the age of 18 days does not reach the level of adult animals (58.6 ± 7.7, 94.5 ± 8.8, and 168.4 ± 11.8 μM/s, respectively). The antagonist of vasopressin V₂ receptors desmopressin at 1 nM significantly increased P _f in both 18-day-old and adult mice but induced no changes in 9-day-old animals. The inhibitor of protein kinase C Ro-31-8220 in the concentration of 100 nM inhibited the desmopressin effect on P _f in 18 day-old and adult mice but did not inhibit the effect of the analog of the vasopressin secondary messenger cAMP, N⁶,O²-dibutyryl cyclic monophosphate, on P _f of the plasma membrane in collecting duct cells. Thus, the response of collecting duct cells to vasopressin appears at the end of wining and correlates with the increase in unstimulated osmotic water permeability of the plasma membrane in collecting duct cells. The vasopressin signal transduction via V₂ receptors is proposed to require the activity of protein kinase C and calcium-dependent systems of intercellular mediators apart from the cAMP-mediated mechanism.     

Metabolic depletion of sphingolipids enhances the mobility of the human serotonin1A receptor

Sphingolipids are essential components of eukaryotic cell membranes. We recently showed that the function of the serotonin_1A receptor is impaired upon metabolic depletion of sphingolipids using fumonisin B₁ (FB₁), a specific inhibitor of ceramide synthase. Serotonin_1A receptors belong to the family of G-protein coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral and developmental functions. Since function and dynamics of membrane receptors are often coupled, we monitored the lateral dynamics of the serotonin_1A receptor utilizing fluorescence recovery after photobleaching (FRAP) under these conditions. Our results show an increase in mobile fraction of the receptor upon sphingolipid depletion, while the diffusion coefficient of the receptor did not exhibit any significant change. These novel results constitute the first report on the effect of sphingolipid depletion on the mobility of the serotonin_1A receptor. Our results assume greater relevance in the broader context of the emerging role of receptor mobility in understanding cellular signaling.     

Determination of δ-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis

Fluorescence recovery after photobleaching (FRAP) is the preferred method for analyzing the lateral mobility of fluorescently-tagged proteins in the plasma membranes (PMs) of live cells. FRAP experiments are described as being easy to perform; however, the analysis of the acquired data can be difficult. The evaluation procedure must be properly combined with the imaging setup of the confocal microscope to provide unbiased results.With the aim of increasing the accuracy of determining the diffusion coefficient (D) and mobile fraction (M_f) of PM proteins, we developed a novel method for FRAP analysis in the equatorial plane of the cell. This method is based on the calculation of photobleaching characteristics, derived from the light intensity profile and optical parameters of the confocal microscope, and on the model of fluorescent molecule diffusion in PM regions outside of the focal plane. Furthermore, cell movement artifacts in the FRAP data are ameliorated by using a region of interest, which is not fixed but instead moves adaptively in coordination with the movement of cells.When this method was used to determine the mobility of the δ-opioid receptor-eYFP in HEK293 cells, a highly significant decrease in receptor mobility was detected in cholesterol-depleted cells. This decrease was fully reversible by the replenishment of cholesterol levels. Our results demonstrate the crucial role played by cholesterol in the dynamic organization of δ-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and M_f values of other PM proteins.     

A cAMP receptor-like G protein-coupled receptor with roles in growth regulation and development

Dictyostelium discoideum uses G protein-mediated signal transduction for many vegetative and developmental functions, suggesting the existence of G protein-coupled receptors (GPCRs) other than the four known cyclic adenosine monophosphate (cAMP) receptors (cAR1-4). Sequences of the cAMP receptors were used to identify Dictyostelium genes encoding cAMP receptor-like proteins, CrlA-C. Limited sequence identity between these putative GPCRs and the cAMP receptors suggests the Crl receptors are unlikely to be receptors for cAMP. The crl genes are expressed at various times during growth and the developmental life cycle. Disruption of individual crl genes did not impair chemotactic responses to folic acid or cAMP or alter cAMP-dependent aggregation. However, crlA⁻ mutants grew to a higher cell density than did wild-type cells and high-copy-number crlA expression vectors were detrimental to cell viability, suggesting that CrlA is a negative regulator of cell growth. In addition, crlA⁻ mutants produce large aggregates with delayed anterior tip formation indicating a role for the CrlA receptor in the development of the anterior prestalk cell region. The scarcity of GFP-expressing crlA⁻ mutants in the anterior prestalk cell region of chimeric organisms supports a cell-autonomous role for the CrlA receptor in prestalk cell differentiation.     

Genetic analysis of the role of G protein-coupled receptor signaling in electrotaxis

Cells display chemotaxis and electrotaxis by migrating directionally in gradients of specific chemicals or electrical potential. Chemotaxis in Dictyostelium discoideum is mediated by G protein–coupled receptors. The unique Gβ is essential for all chemotactic responses, although different chemoattractants use different receptors and Gα subunits. Dictyostelium amoebae show striking electrotaxis in an applied direct current electric field. Perhaps electrotaxis and chemotaxis share similar signaling mechanisms? Null mutation of Gβ and cAMP receptor 1 and Gα2 did not abolish electrotaxis, although Gβ-null mutations showed suppressed electrotaxis. By contrast, G protein signaling plays an essential role in chemotaxis. G protein–coupled receptor signaling was monitored with PHcrac–green fluorescent protein, which translocates to inositol phospholipids at the leading edge of cells during chemotaxis. There was no intracellular gradient of this protein during electrotaxis. However, F-actin was polymerized at the leading edge of cells during electrotaxis. We conclude that reception and transduction of the electrotaxis signal are largely independent of G protein–coupled receptor signaling and that the pathways driving chemotaxis and electrotaxis intersect downstream of heterotrimeric G proteins to invoke cytoskeletal elements.     

Go contributes to olfactory reception in Drosophila melanogaster

Background  

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology and function as ligand-gated cation channels. Consequently, the involvement of cyclic nucleotides and G proteins in insect odor reception is controversial. Since the heterotrimeric G_oα subunit is expressed in Drosophila olfactory receptor neurons, we reasoned that G_o acts together with insect odorant receptor cation channels to mediate odor-induced physiological responses.     

Molecular neurobiology and pharmacology of the Vasopressin/Oxytocin receptor family

Summary 1. VP and OT mediate their wealth of effects via 4 receptor subtypes V_1a, V_1b, V₂, and OT receptors.2. We here review recent insights in the pharmacological properties, structure activity relationships, species differences in ligand specificity, expression patterns, and signal transduction of VP/OT receptor.3. Furthermore, the existence of additional VP/OT receptor subtypes is discussed.     

Guanine nucleotides modulate the function of chemotactic cyclic AMP receptors inDictyostelium discoideum

Summary Guanosine di- and triphosphates specifically decrease the affinity of chemotactic cAMP receptors in isolatedDictyostelium discoideum membranes. The K_0.5 was increased from 50 nM to 150 nM. Receptors were shown to be heterogeneous in dissociation kinetics. In the absence of guanine nucleotides three dissociation processes could be resolved, having first order rate constants of 8.7 x 10⁻⁴, 1.3 X 10⁻², and higher than 0.1 s⁻¹. Guanine nucleotides decreased the affinity for cAMP by transforming the slowest dissociating receptor form (K_D is 8 nM) to forms dissociating more rapidly. Our data indicate that a guanine nucleotide binding protein (G-protein) is involved in the transduction of the cAMP signal inD. discoideum.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4

Triiodothyronine (T₃) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T₃/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T₃ at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T₃-mediated regulation of DKK4 remains unknown. In the present study, the 5′ promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T₃ response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides −1645 and −1629 conferring T₃ responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T₃/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.     

Heterodimerization of Mouse Orexin type 2 receptor variants and the effects on signal transduction

Orexin-A and Orexin-B play important roles in many physiological processes in which Orexins orchestrate diverse downstream effects via two G-protein coupled receptors: Orexin1R and Orexin2R. Two alternative C-terminus splice variants of the mouse Orexin receptors mOX2αR and mOX2βR have recently been identified. This study explored the possibility of heterodimerization between mOX2αR and mOX2βR, and investigated novel signal transduction characteristics after stimulation. The dimerization of mOX2αR and mOX2βR was confirmed by BRET and co-immunoprecipitation assays. Meanwhile, in HEK293 cells, co-expression of mOX2αR and mOX2βR resulted in a strengthened increase in activation of ERK1/2, with maximal activation at 5 min and 100 nM. Furthermore, heterodimerization also elicits stronger intracellular Ca^2 + elevation after Orexin(s) stimulation, followed by a slower decline in intracellular Ca^2 + to a steady endpoint. Protein Kinase C Inhibitor significantly inhibited these downstream effects. In addition, the cAMP response element reporter activities were significantly reduced, whereas the serum response element luciferase and the T-lymphocyte activation of nuclear factor-responsive element reporter activity were significantly up-regulated after Orexin(s) stimulation. Besides, Orexin-A/-B induced a significantly higher rate of HEK293 cell proliferation in cells co-expressing mOX2αR/mOX2βR compared to the control group. Taken together, we provide conclusive evidence that mOX2αR can form a functional heterodimer with mOX2βR and this leads to increased PKC and decreased protein kinase A activity by ERK signal pathway leading to a significant increase in cell proliferation. The nature of this signaling pathway has significant implications for the role of Orexin in the regulation of physiological processes including the homeostasis of feeding.     

Peculiarities of Signal Transduction from Arginine-Vasopressin Receptors Located on the Apical Surface of Epithelial Cells of the Frog Urinary Bladder and on the Amoeba Outer Membrane

Mechanisms of action of arginine-vasopressin (AVP) on water transport across the wall of urinary bladder of the frog R. temporaria L. and the outer membrane of the amoeba A. proteus were considered. AVP and its functional analogs were added to the basolateral and apical surfaces of the frog urinary bladder membrane and to the amoeba outer surface. In amoeba the AVP effect was evaluated from action on frequency of contractions of contractile vacuole that represents its organ of water—electrolyte homeostasis. It was shown that the system of signal transduction from the apical AVP receptors differed considerably from that located on the basolateral membrane and had a marked similarity with the system of signal transduction from AVP-sensitive structures on the outer membrane of amoeba. The urinary bladder apical membrane as well as the amoeba outer membrane has a mixed type of sensitivity to AVP with unidirectional (not antagonistic like on the basolateral membrane) involvement of systems coupled to receptors of the V₁ and V₂ type. We suggest that the system of signal transduction from apical AVP receptors corresponds to the earlier stage of evolutionary development.