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. Receptor 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 vasopressiin V₂-receptor is highly laterally mobile at 37°C. It has recently been possible to reversibly modulate the V₂-receptor mobile fraction (F) to largely varuing extents and to demonstrate thereby a direct effect on the maximal rate of in vivo cAMP production at 37°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-K_D (physiological) hormone concentrations, with mobile receptors being required to effect G-protein activation.     

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.     

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     

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.     

Structural requirements for V2 vasopressin receptor proteolytic cleavage.

The ligand-induced proteolytic cleavage of the V2 vasopressin receptor transiently expressed in COS cells was investigated. After incubation of the cell membranes with a photoreactive ligand possessing full agonistic properties for V2 receptors, approximately 90% of the porcine and bovine V2 vasopressin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptors. The oxytocin receptor was completely resistant to proteolysis after binding the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V2/oxytocin receptors obtained by transfer of extracellular domains of the oxytocin receptor into the V2 receptor showed an increase in binding affinity for oxytocin versus vasopressin and a diminished cleavage. The proteolysis-resistant chimeric V2/oxytocin receptor, which contains the first three extracellular domains of the oxytocin receptor, stimulated cAMP accumulation to a larger extent in response to vasopressin than the wild-type receptor and showed impaired desensitization of the adenylate cyclase system. Our data indicate that the proteolytic cleavage of the V2 receptor requires a defined conformation, especially of the first two extracellular domains that is induced by agonist binding. Furthermore, the results suggest that the proteolytic V2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.     

Aberrant receptor-mediated Cushing's syndrome

Multiple alterations of G-protein-coupled receptors and G-proteins regulating intracellular transduction signal have been described in endocrine tumours. In Cushing's syndrome, aberrant or 'illicit' expression of membrane receptors (mainly G-protein-coupled receptors) has been observed in adrenal adenomas and adrenocorticotropic hormone (ACTH)-independent macronodular bilateral adrenal hyperplasia. The best characterized example to date is the aberrant expression of the gastric inhibitory polypeptide receptor that causes 'food-dependent hypercortisolism'. Aberrant expression of the luteinizing hormone, 2-adrenergic, interleukin receptors have also been reported. The level of expression of the vasopressin V1a receptor correlates with the direct (ACTH-independent) cortisol response to vasopressin.     

Desensitization of vasopressin sensitive adenylate cyclase by vasopressin and phorbol esters

Desensitization of vasopressin V2 receptor-mediated adenylate cyclase was studied in canine kidney cell line, MDCK cells. Overnight treatment of MDCK cells with arginine vasopressin (AVP) resulted in a loss of vasopressin receptors and an inhibition of cAMP accumulation in response to AVP. Both the loss of receptor and reduction in cAMP accumulation were time- and AVP concentration-dependent. Desensitization was selective for AVP because cAMP formation in response to isoproterenol, prostaglandin E1 (PGE1) and forskolin was not affected by AVP pre-treatment. Pre-treatment of MDCK cells with phorbol dibutyrate (PDBu) also caused a dose-dependent inhibition of AVP mediated cAMP accumulation, but not of isoproterenol-, PGE1- and forskolin-induced cAMP accumulation. PDBu pre-treatment did not cause loss of vasopressin receptors. Instead, the affinity for vasopressin was changed by PDBu treatment. Pre-treatment of the cells with pertussis toxin (PT) had no effect on the desensitization and downregulation of vasopressin (V2) receptors, suggesting that the desensitization may not be mediated by pertussis toxin sensitive G-protein. Our data suggest that pre-treatment of MDCK cells with AVP or PDBu caused desensitization of AVP-mediated cAMP accumulation and that downregulation of V2 receptors required agonist occupancy of the receptors, whereas the affinity of the receptors was changed by phorbol ester treatment.     

Insulin processing and signal transduction in rat adipocytes

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.     

Relationship of (8-lysine) vasopressin receptor transition to receptor functional properties in a pig kidney cell line (LLC-PK1)

In intact LLC-PK1 cells, occupancy of vasopressin receptors (Roy, C., and Ausiello, D. A. (1981) J. Biol. Chem. 256, 3415-3522) correlated with cell cAMP production. This relationship was observed as a function of hormone dose, incubation time, and changes in receptor affinity. However, the rate of cAMP production diminished with time in intact cells exposed to high hormone concentrations, even in the presence of a phosphodiesterase inhibitor. A rapid desensitization of adenylate cyclase activity was observed in minutes upon treatment of intact cells with high hormonal concentrations. Desensitization was dose- and time-dependent. Hypertonic sodium chloride, which increased hormonal binding and cell cAMP production, prevented desensitization. The acute decrease in hormone-stimulated adenylate cyclase activity correlated with increased occupancy of low affinity binding sites. EDTA-suspended cells, which have a homogeneous population of binding sites, did not demonstrate desensitization. A proposal is made as to the consequences of this phenomenon at physiological concentrations of vasopressin.     

Protein kinase C modulates effects of prostanoids on cyclic adenosine monophosphate in guinea pig chief cells

In the course of examining the role of protein kinase C in signal transduction in dispersed chief cells from guinea pig stomach, we observed that phorbol esters inhibit prostaglandin (PG)-stimulated increases in cyclic adenosine monophosphate (cAMP). Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, decreased maximal levels of PGE2-stimulated cAMP by 40%. This dose-dependent effect was observed within 30 sec and was maximal by 1 min of incubation at 37 degrees C. Phorbols that do not activate protein kinase C did not have this effect. Adding H7, a protein kinase C inhibitor, abolished the inhibitory effects of PMA, indicating that these effects are not caused by activation of cyclic nucleotide phosphodiesterases. PMA did not alter the increase in cellular cAMP caused by cholera toxin, forskolin, secretin, or vasoactive intestinal peptide. Hence the site of these prostanoid-specific actions of protein kinase C does not appear to be stimulatory or inhibitory guanine nucleotide binding proteins or the catalytic component of the adenylyl cyclase system. In dispersed chief cells, activation of protein kinase C may inhibit prostanoid-induced stimulation of the adenylyl cyclase system by a direct effect on prostaglandin receptors.     

Electric field-induced redistribution and postfield relaxation of epidermal growth factor receptors on A431 cells

The lateral mobility of the epidermal growth factor (EGF) receptor in the plane of the plasma membrane of cultured A431 cells was investigated using direct and indirect fluorescent probes to measure the generation and relaxation of electric field-induced receptor asymmetry. A steady electric field of 15 V/cm for 30 min at 23 degrees C induced a redistribution of the unoccupied EGF receptor such that there was approximately a three-fold higher concentration of receptors at the cathode-facing pole. After termination of the field, the unoccupied receptors back diffused at 37 degrees C with a rate corresponding to a diffusion coefficient of 2.6-3.5 X 10(-10) cm2/s. No diffusion was detected at 4 degrees C. Formation of the hormone-receptor complex is known to induce receptor clustering and internalization. By inhibiting internalization with metabolic poisons, we were able to study the cell surface mobility of clusters of the hormone-receptor complex. The same degree of asymmetry was induced when the occupied receptor was exposed to an electric field and the rate of back diffusion of clusters of the hormone-receptor complex corresponded to a diffusion coefficient of 0.68-0.95 X 10(-10) cm2/s. Although the unoccupied receptor is somewhat more mobile than the hormone-receptor complex, it was still far less mobile than one would predict for an unconstrained protein imbedded in a phospholipid bilayer.     

Cross talk between stimulatory and inhibitory guanosine 5'-triphosphate binding proteins: role in activation and desensitization of the adenylate cyclase response to vasopressin

The inhibitory GTP-binding protein (Gi) is known to mediate the effects of a number of hormones that act through specific receptors to inhibit adenylate cyclase. In this study we examined the mechanism whereby Gi modulates the response of adenylate cyclase to a stimulatory hormone and its role in desensitization. In membranes prepared from the cultured renal epithelial cell line LLCPK1, adenylate cyclase activity was stimulated 16-fold by 1-2 microM lysine vasopressin. Addition of GTP (1-100 microM) resulted in stimulation of basal activity but inhibition of hormone-stimulated activity (approximately 40% inhibition at 100 microM GTP). This contrasts with the usual effect of GTP to support or augment activation by stimulatory receptors. The inhibitory effect was abolished by pertussis toxin, which had little effect on basal activity in the absence or presence of added GTP or on vasopressin-stimulated activity in the absence of added GTP. GTP-mediated inhibition was vasopressin concentration dependent. At concentrations of vasopressin below the K1/2 for enzyme activation (approximately 0.6 nM), GTP was stimulatory, and at higher concentrations, GTP was inhibitory. The inhibitory effect of GTP was also observed for a V2-receptor agonist and was not abolished by a V1-receptor antagonist, indicating that a distinct V1 receptor did not mediate inhibition of adenylate cyclase. Using the known subunit structure of adenylate cyclase, we developed the minimal mechanism that would incorporate a modulatory role for Gi in determining net activation of adenylate cyclase by a stimulatory hormone. The predicted enzyme activities for basal and maximal hormone stimulation in the presence and absence of GTP were generated, and model parameters were chosen to match the experimental observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

An arginyl in the N-terminus of the V1a vasopressin receptor is part of the conformational switch controlling activation by agonist.

Defining how the agonist-receptor interaction differs from that of the antagonist-receptor and understanding the mechanisms of receptor activation are fundamental issues in cell signalling. The V1a vasopressin receptor (V1aR) is a member of a family of related G-protein coupled receptors that are activated by neurohypophysial peptide hormones, including vasopressin (AVP). It has recently been reported that an arginyl in the distal N-terminus of the V1aR is critical for binding agonists but not antagonists. To determine specific features required at this locus to support high affinity agonist binding and second messenger generation, Arg46 was substituted by all other 19 encoded amino acids. Our data establish that there is an absolute requirement for arginyl, as none of the [R46X]V1aR mutant constructs supported high affinity agonist binding and all 19 had defective signalling. In contrast, all of the mutant receptors possessed wildtype binding for both peptide and nonpeptide antagonists. The ratio of Ki to EC50, an indicator of efficacy, was increased for all substitutions. Consequently, although [R46X]V1aR constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype V1aR to become activated. Therefore, in the wildtype V1aR, Arg46 constrains the inactive conformation of the receptor. On binding AVP this constraint is alleviated, promoting the transition to active V1aR. Our findings explain why arginyl is conserved at this locus throughout the evolutionary lineage of the neurohypophysial peptide hormone receptor family of G-protein coupled receptors.     

Identification of a novel epitope in the thyroid-stimulating hormone receptor ectodomain acting as intramolecular signaling interface

Glycoprotein hormone receptors (GPHRs) differ from the other seven transmembrane receptors mainly through a complex activation mechanism that requires the binding of a large hormone toward a large N-terminal ectodomain. The intramolecular mechanism of the signal transduction to the serpentine domain upon hormone binding at the ectodomain is not understood. To identify determinants at the GPHR ectodomain that may be involved in signal transduction, we first searched for homologous structural features. Based on high sequence similarity to the determined structures of the Nogo-receptor ectodomain and the intermolecular complex of the Interleukin-8 ligand (IL8) and the N-terminal peptide of the IL8 receptor (IL8RA), the hypothesis was developed that portions of the intramolecular components, Cysteine-box-2 and Cysteine-box-3, of the GPHR ectodomain interact and localize at the interface between ectodomain and serpentine domain. Indeed, point mutations within the D403EFN406 motif at Cysteine-box-3 of the thyrotropin receptor resulted in increased basal cAMP levels, suggesting that this motif may be important for transduction of the signal from the ectodomain to the transmembrane domain. New indications are provided about the tight spatial cooperation and relative location of the new epitope and other determinants at the thyrotropin receptor ectodomain, such as the leucine-rich repeat motif Ser281 and the cysteine boxes. According to the high sequence conservation, the results are of general relevance for the signal transduction mechanism of other glycoprotein hormone receptors such as choriogonadotrophic/luteinizing hormone receptor and follicle-stimulating hormone receptor.     

Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells

The dynamic process of embryonic cell motility was investigated by analyzing the lateral mobility of the fibronectin receptor in various locomotory or stationary avian embryonic cells, using the technique of fluorescence recovery after photobleaching. The lateral mobility of fibronectin receptors, labeled by a monoclonal antibody, was defined by the diffusion coefficient and mobile fraction of these receptors. Even though the lateral diffusion coefficient did not vary appreciably (2 X 10(-10) cm2/S less than or equal to D less than or equal to 4 X 10(-10) cm2/S) with the locomotory state and the cell type, the mobile fraction was highly dependent on the degree of cell motility. In locomoting cells, the population of fibronectin receptors, which was uniformly distributed on the cell surface, displayed a high mobile fraction of 66 +/- 19% at 25 degrees C (82 +/- 14% at 37 degrees C). In contrast, in nonmotile cells, the population of receptors was concentrated in focal contacts and fibrillar streaks associated with microfilament bundles and, in these sites, the mobile fraction was small (16 +/- 8%). When cells were in a stage intermediate between highly motile and stationary, the population of fibronectin receptors was distributed both in focal contacts with a small mobile fraction and in a diffuse pattern with a reduced mobile fraction (33 +/- 9%) relative to the diffuse population in highly locomotory cells. The mobile fraction of the fibronectin receptor was found to be temperature dependent in locomoting but not in stationary cells. The mobile fraction could be modulated by affecting the interaction between the receptor and the substratum. The strength of this interaction could be increased by growing cells on a substratum coated with polyclonal antibodies to the receptor. This caused the mobile fraction to decrease. The interaction could be decreased by using a probe, monoclonal antibodies to the receptor known to perturb the adhesion of certain cell types which caused the mobile fraction to increase. From these results, we conclude that in locomoting embryonic cells, most fibronectin receptors can readily diffuse in the plane of the membrane. This degree of lateral mobility may be correlated to the labile adhesions to the substratum presumably required for high motility. In contrast, fibronectin receptors in stationary cells are immobilized in focal contacts and fibrillar streaks which are in close association with both extracellular and cytoskeletal structures; these stable complexes appear to provide firm anchorage to the substratum.     

Luteinizing hormone receptors are self-associated in slowly diffusing complexes during receptor desensitization

We have previously shown that rat LH receptors (LHRs) occupied by human CG (hCG) exhibit slow receptor lateral diffusion and are self-associated. Here we have examined whether LHRs become self-associated and enter slowly diffusing structures in response to hormone binding and whether these receptors retain this organization while in the desensitized state. Before hormone exposure, wild-type rat LHRs coupled at the C terminus to enhanced green fluorescent protein (GFP-LHR-wt) exhibited fast lateral diffusion, as assessed by fluorescent photobleaching recovery (FPR) methods, and most receptors were laterally mobile. After 30 min exposure to hCG and subsequent removal of hormone by low pH wash, hormone challenge at any time within the next 4 h produced no increase in cellular cAMP levels. During this time, LHRs were either laterally immobile or exhibited slower lateral diffusion. When LHRs were again responsive to binding of hormone, the rate of receptor lateral diffusion had become significantly faster and the fraction of mobile receptors was again large. Desensitized LHRs were also self-associated and present in microscopically visible clusters on the plasma membrane. Fluorescence energy transfer (FET) methods were used to measure the extent of interaction between receptors coupled to either GFP or to yellow fluorescent protein (YFP). Before hormone treatment, there was essentially no energy transfer between LHRs. After desensitization of the receptors by 30 min exposure to hCG, energy transfer efficiency increased to 18%. Values for FET efficiency between desensitized receptors decreased over time, and receptors were responsive to hormone only after measurable energy transfer had completely disappeared. Together these results suggest that desensitized LHRs exist in large, slowly diffusing structures containing self-associated receptors and that these structures must dissipate before the receptor can again respond to hormone.     

Ammonium chloride affects receptor number and lateral mobility of the vasopressin V2-type receptor in the plasma membrane of LLC-PK1 renal epithelial cells: role of the cytoskeleton

The acidotropic agent ammonium chloride (NH4Cl) not only affects receptor metabolism by inhibiting lysosomal acidification, but can also affect the targeting of proteins to specific membranes in polarized cells, possibly through effects mediated by the cytoskeleton. The present study examines the effects of NH4Cl and perturbers of cytoskeleton structure on vasopressin V2 receptor expression in LLC-PK1 renal epithelial cells. Surprisingly, long-term pretreatment of cells with NH4Cl or short-term treatment with the actin perturber cytochalasin B resulted in an up to 70% increase in specific Arg-8-vasopressin binding compared to control cells, which was independent of the presence of NH4Cl in the binding test, and apparently the result of increased V2 receptor expression. Perturbers of microtubules such as colchicine and vinblastine had no such effect. A rhodamine-labeled analog of vasopressin was used to fluorescently label the V2 receptor of LLC-PK1 cells, and microscopic measurements of membrane-localized fluorescence confirmed the increased V2 receptor expression in the basal plasma membrane subsequent to NH4Cl pretreatment. Lateral mobility of the V2 receptor was measured in living cells using the technique of microphotolysis (photobleaching). The fraction of mobile receptors was 0.2 in cells pretreated with NH4Cl, markedly reduced compared to that of 0.9 in untreated cells. The apparent lateral diffusion coefficient D was about 3 x 10(-10) cm2/s in both pretreated and untreated cells. Results for fluorescence labeling of the actin cytoskeleton indicate that NH4Cl pretreatment of LLC-PK1 cells results in perturbation of microfilament structure. All results imply that the cytoskeleton plays a central role in V2 receptor expression and lateral mobility.