Receptor-ligand Dissociation Research Articles

The V-ATPase a2-subunit as a putative endosomal pH-sensor

V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

Fluid mechanical and physicochemical modeling interprets hypertension to be capable of inducing secondary complications

The relationship of hypertensives and many pathological syndromes still remains unclear. A mathematical model in terms of the fluid mechanics and physicochemical analyses is established to correlate the plasma viscosity, the shear stress and the rate of shear in blood stream with the ligand-receptor dissociation constant. This model has arrived at the conclusive results that high viscosity, high rate of shear created in the blood streams, and the peripheral resistance may act as important preceding factors to induce a serial subsequent pathological clinical manifestations. High viscosity may interfere with the ligand-receptor combination, in contrast, high rate of shear may knock the ligand (s) off the existing ligand-receptor complex, while elevation of peripheral resistance may slow down the blood flow rate, resulting in a diminished dissociation of ligand-receptor complex. This model has successfully interpreted the possible cause of some post-hypertensive abnormal outcome manifestations involving obstructive and degenerative stenosis (such as renal artery stenosis), growth retardation, blood vessel detriment, coarctation of aorta, coronary thrombotics, atherosclerosis, hyperinsulinemia, diabetes, obesity, hypothyroidism, infertility, and at the worst, carcinoma, etc.

Bistability of Cell-Matrix Adhesions Resulting from Nonlinear Receptor-Ligand Dynamics

Bistability is a major mechanism for cellular decision making and usually results from positive feedback in biochemical control systems. Here we show theoretically that bistability between unbound and bound states of adhesion clusters results from positive feedback mediated by structural rather than biochemical processes, namely by receptor-ligand dissociation and association dynamics that depend nonlinearly on mechanical force and receptor-ligand separation. For small cell-matrix adhesions, we find rapid switching between unbound and bound states, which in the initial stages of adhesion allows the cell to explore its environment through many transient adhesions.

Inhibition of sequestration of human B2 bradykinin receptor by phenylarsine oxide or sucrose allows determination of a receptor affinity shift and ligand dissociation in intact cells

Depending on their interaction with intracellular proteins, G protein-coupled receptors (GPCR) often display different affinities for agonists at 37 degrees C. Determining the affinity at that temperature is often difficult in intact cells as most GPCRs are internalized after activation. When sequestration of the B2 bradykinin receptor (B2R) was inhibited by either 0.5 M sucrose or phenylarsine oxide (PAO), a shift in the affinity was detected when the incubation temperature was raised from 4 degrees C to 37 degrees C or lowered from 37 degrees C to 4 degrees C. In contrast, binding of the antagonist [3H]NPC 17731 was temperature-independent. B2R mutants displayed different affinity shifts allowing conclusions on the role of the involved amino acids. By inhibiting receptor sequestration it was possible to determine also dissociation of [3H]BK and of [3H]NPC 17731 from intact cells at 37 degrees C. Surprisingly, both dissociation rates were markedly enhanced by the addition of unlabeled ligand, most likely via prevention of reassociation of dissociated [3H]ligand. This suggests that dissociated [3H]ligand cannot move freely away from the receptor. In summary, our data demonstrate that inhibition of receptor internalization either by PAO or sucrose provides an excellent method to study receptor function and the effects of mutations in intact cells.

Receptor-mediated endocytosis by the asialoglycoprotein receptor: effect of ethanol administration on endosomal distribution of receptor and ligand.

Using the asialoglycoprotein receptor (ASGP-R) and a representative ligand, asialoorosomucoid (ASOR), we have previously shown ethanol-induced impairment of endosomal acidification, receptor recycling and ligand binding, internalization, and degradation. In the current study, we further investigated ethanol-induced alterations in receptor/ligand trafficking by labeling endosomes in vivo with either Texas-Red-ASOR or 125I-ASOR, and then assessing the receptor/ligand content of endosomes. We assessed two fractions after both 5 and 25 min of labeling: 'early endosomes' (EEs; endosomes from the cell periphery) and 'late endosomes' (LEs; endosomes farther into the cell interior). At both time points, significantly more ligand was found in EE fractions isolated from chow- and pair-fed controls (3:1, EE to LE, respectively). However, endosomes isolated from ethanol-fed animals showed a shift over time toward a more equal ligand distribution between endosome fractions (P < or = 0.05). Analysis of the ASGP-R content revealed a distribution pattern between the endosome fractions similar to that observed for ligand distribution. Impairment of receptor-ligand dissociation was assessed in endosome fractions by determining bound/free ligand ratios. Analysis showed that most of the ligand present in both endosome fractions was free (56-99%), although more was bound to receptor in EE vs LE of both control and ethanol animals (P < or = 0.05). At 5 min, more ligand remained bound in endosomes from ethanol-fed animals compared with control endosomes (P < or = 0.05), and the same pattern was observed at the latter time point. These results suggest that delayed dissociation may cause the receptor ligand complexes to travel farther into the cell interior, which may impair proper trafficking of the ligand to lysosomes and alter the receptor recycling.

Cooperative adhesion of ligand–receptor bonds

Cooperative (simultaneous) breakage of multiple adhesive bonds has been proposed as a mechanism for enhanced binding strength between adhesion molecules on apposing cell surfaces. In this report, we used the atomic force microscopy (AFM) to study how changes in binding affinity and separation rate of force-induced ligand–receptor dissociation affect binding cooperativity. The AFM force measurements were carried out using (strept)avidin-functionalized cantilever tips and biotinylated agarose beads under conditions where multiple (strept)avidin–biotin linkages were formed following surface contact. At slow surface separation of the AFM cantilever from the bead's surface, the (strept)avidin–biotin linkages appeared to rupture sequentially. Increasing the separation rate from 210 to 1950 nm/s led to a linear increase in the average rupture force. Moreover, force histograms revealed a quantized force distribution that shifted toward higher values with increasing separation rate. In measurements of streptavidin–iminobiotin adhesion, the force distribution also shifted toward higher values when the buffer was adjusted to a higher pH to raise the binding affinity. Together, these results demonstrate that the cooperativity of ligand–receptor bonds is significantly enhanced by increases in surface separation rate and/or binding affinity.

Lateral mobility and specific binding to GABA(A) receptors on hippocampal neurons monitored by fluorescence correlation spectroscopy.

The binding behavior of a fluorescently labeled muscimol derivative to the GABA(A) receptor was analyzed at rat hippocampal neurons by fluorescence correlation spectroscopy. After muscimol had been labeled with the fluorophore Alexa Fluor 532, specific binding constants for binding of the dye-labeled ligand (Mu-Alexa) to the GABA(A) receptor were determined. We found a high specific binding affinity of Mu-Alexa with a K(D) value of 3.4 +/- 0.5 nM and a rate constant of ligand-receptor dissociation (k(diss)) of (5.37 +/- 0.95) x 10(-2) s(-1). A rate constant of ligand-receptor association (k(ass)) of (1.57 +/- 0.28) x 10(7) L mol(-1) s(-1) was calculated. The following diffusion coefficients were observed: D(free) = 233 +/- 20 microm(2)/s (n = 66) for free diffusing Mu-Alexa, D(bound1) = 2.8 +/- 0.9 microm(2)/s (n = 64) for the lateral mobility, and D(bound2) = 0.14 +/- 0.05 microm(2)/s (n = 56) for the hindered mobility of the GABA(A) receptor-ligand complex in the cell membrane. Saturation of Mu-Alexa binding was observed at a concentration of 50 nM. A maximum number of binding sites [B(max) = 18.4 +/- -0.4 nM (n = 5)] was found. Similar K(i) values of 4.5 +/- 1.0 nM for nonlabeled muscimol and 8.8 +/- 1.8 nM for Mu-Alexa were found by RRAs using [(3)H]muscimol as a radioligand. A concentration-dependent increase in the level of specific Mu-Alexa binding was demonstrated by the positive cooperative activity of co-incubated midazolam, which was selectively found in GABA(A) receptor-ligand complexes with hindered mobility.

Benzodiazepine binding studies on living cells: application of small ligands for fluorescence correlation spectroscopy.

We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex.

Molecular and Cellular Physiology of the Dissociation of Atrial Natriuretic Peptide from Guanylyl Cyclase A Receptors

Guanylyl cyclase subtype A (GCA) is the main receptor that mediates the effects of atrial natriuretic peptide (ANP) in the regulation of plasma volume and blood pressure. The dynamics of the dissociation of ANP from GCA were investigated in cultured Chinese hamster ovary (CHO) cells stably transfected with wild-type (WT) or mutant GCA receptors. The rate of dissociation of specifically bound (125)I-ANP-(1-28) from intact CHOGCAWT cells at 37 degrees C was extremely rapid (K(off) = 0.49 +/- 0.02 min(-1)), whereas in isolated membranes prepared from these cells, the dissociation at 37 degrees C was >10-fold slower (K(off) = 0.035 +/- 0.006 min(-1)). The dissociation of ANP from CHOGCAWT cells showed remarkable temperature dependence. Between 22 and 37 degrees C, K(off) increased approximately 8 times, whereas between 4 and 22 degrees C, it increased only 1.5 times. Total deletion of the cytoplasmic domain or of the catalytic guanylyl cyclase sequence within this domain abolished ANP-induced increases in cGMP, dramatically slowed receptor-ligand dissociation by at least 10-fold, and abolished the temperature dependence of the dissociation of ANP. Deletion of the kinase-like domain led to maximal constitutive activation of guanylyl cyclase, markedly decreased K(off) to 0.064 +/- 0.006 min(-1), and also abolished the temperature dependence of dissociation. Substitution of Ser(506) by Ala and particularly the double substitution of Gly(505) and Ser(506) by Ala within the kinase-like domain markedly reduced ANP-induced increases in cGMP, whereas K(off) decreased modestly (albeit significantly) to 0.36 +/- 0.03 and 0.24 +/- 0.02 min(-1), respectively. As a whole, the results demonstrate for the first time that temperature per se or ATP alone cannot account for rapid GCA receptor-ligand dissociation under physiological conditions and suggest that ligand dissociation is modulated in part by the interaction of still unidentified cytosolic factors with the cytoplasmic domain of GCA.

Kinetic proofreading models for cell signaling predict ways to escape kinetic proofreading.

In the context of cell signaling, kinetic proofreading was introduced to explain how cells can discriminate among ligands based on a kinetic parameter, the ligand-receptor dissociation rate constant. In the kinetic proofreading model of cell signaling, responses occur only when a bound receptor undergoes a complete series of modifications. If the ligand dissociates prematurely, the receptor returns to its basal state and signaling is frustrated. We extend the model to deal with systems where aggregation of receptors is essential to signal transduction, and present a version of the model for systems where signaling depends on an extrinsic kinase. We also investigate the kinetics of signaling molecules, "messengers," that are generated by aggregated receptors but do not remain associated with the receptor complex. We show that the extended model predicts modes of signaling that exhibit kinetic discrimination for some range of parameters but for other parameter values show little or no discrimination and thus escape kinetic proofreading. We compare model predictions with experimental data.

Cadmium Inhibits Albumin Endocytosis in Opossum Kidney Epithelial Cells

Chronic exposure to cadmium results in proteinuria. To gain insights into the mechanism by which cadmium inhibits the protein transport in the renal proximal tubule, we investigated the effects of cadmium on the receptor-mediated endocytosis of albumin, using fluorescein isothiocyanate-labeled bovine serum albumin (FITC-albumin) as a model substrate and opossum kidney cell line (OK cell) as a proximal tubular cell model. Cell monolayers grown to confluence were treated with 100 μM CdCl2 for 60 min at 37°C, washed, and tested for FITC-albumin uptake (37°C) and surface binding (4°C). The amounts of FITC-albumin uptake and binding were quantified by fluorimetrically determining the cell-adherent fluorescence. Both the binding and uptake of FITC-albumin by OK cells appeared to be saturable and inhibitable by unlabeled albumin in the medium, indicating that specific receptor sites were involved. The uptake of FITC-albumin was inhibited by agents that interfere with the formation of endocytotic vesicle (hypertonic mannitol), endosomal acidification (NH4Cl), and vesicular trafficking (cytochalasin D and nocodazole), confirming that the uptake occurred via the process of receptor-mediated endocytosis. In cells treated with cadmium, the specific FITC-albumin uptake was significantly attenuated, and this was due to a reduction in Vmax and a rise in Km. These changes in kinetic parameters were similar to those induced by NH4Cl. The binding of FITC-albumin to the apical surface of OK cells was inhibited by cadmium treatment, and this was attributed to a reduction in Bmax. The values of Kd and its pH dependency were not altered by cadmium treatment. The formation of endocytotic vesicles, as judged by fluid phase endocytosis of FITC-inulin, was not changed by cadmium treatment. These results indicate that the receptor-mediated endocytosis of albumin is impaired in cadmium-treated OK cells most likely due to a defect in endosomal acidification and the attendant fall in ligand-receptor dissociation, which impairs receptor recycling and the overall efficiency of endocytosis.

Inhibition of Na+-H+ exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum.

1. Receptor-mediated endocytosis is an important mechanism for transport of macromolecules and regulation of cell-surface receptor expression. In renal proximal tubules, receptor-mediated endocytosis mediates the reabsorption of filtered albumin. Acidification of the endocytic compartments is essential because it interferes with ligand-receptor dissociation, vesicle trafficking, fusion events and coat formation. 2. Here we show that the activity of Na+-H+ exchanger isoform 3 (NHE3) is important for proper receptor-mediated endocytosis of albumin and endosomal pH homeostasis in a renal proximal tubular cell line (opossum kidney cells) which expresses NHE3 only. 3. Depending on their inhibitory potency with respect to NHE3 and their lipophilicity, the NHE inhibitors EIPA, amiloride and HOE694 differentially reduced albumin endocytosis. The hydrophilic inhibitor HOE642 had no effect. 4. Inhibition of NHE3 led to an alkalinization of early endosomes and to an acidification of the cytoplasm, indicating that Na+-H+ exchange contributes to the acidification of the early endosomal compartment due to the existence of a sufficient Na+ gradient across the endosomal membrane. 5. Exclusive acidification of the cytoplasm with propionic acid or by removal of Na+ induced a significantly smaller reduction in endocytosis than that induced by inhibition of Na+-H+ exchange. 6. Analysis of the inhibitory profiles indicates that in early endosomes and endocytic vesicles NHE3 is of major importance, whereas plasma membrane NHE3 plays a minor role. 7. Thus, NHE3-mediated acidification along the first part of the endocytic pathway plays an important role in receptor-mediated endocytosis. Furthermore, the involvement of NHE3 offers new ways to explain the regulation of receptor-mediated endocytosis.

Differential Effects of Monensin on Asialoglycoprotein Receptor Function after Short-Term Ethanol Administration

Chronic ethanol consumption is associated with multiple impairments in receptor-mediated endocytosis (RME) by the hepatic asialoglycoprotein receptor (ASGP-R). Previous work on this receptor has shown that its activity can be perturbed by the carboxylic ionophore monensin. This agent has been shown to preferentially affect receptor–ligand dissociation and receptor redistribution of one subset (State 2) of ASGP-R, while receptor function in a second subset (State 1 receptors) is unaffected. In the present study, we examined the effect of monensin on ASGP-R activity and intracellular receptor–ligand dissociation after 7–10 days of ethanol feeding, a time when we have shown altered ASGP-R function in ethanol-fed animals. Hepatocytes from male Wistar rats (fed an ethanol-containing or control diet) were utilized. Ethanol administration decreased total ligand binding by 35–40% ( P < 0.01) without a change in receptor protein content. After monensin treatment, surface receptors on cells from control animals were inactivated and redistributed to the cell interior. In cells from ethanol-fed animals, a similar pattern of monensin-induced inactivation was shown, but no redistribution occurred. Intracellular receptor–ligand dissociation was impaired in both cell types, although the monensin-induced effect on dissociation was significantly less dramatic (two-fold) in the hepatocytes from ethanol-fed animals as compared with controls. Thus, although receptors on both cell types were susceptible to monensin, cells from the ethanol-fed animals were less vulnerable to the added effects of this agent. Since monensin affects functioning of State 2, but not State 1 receptors, a very early effect of ethanol may be a preferential impairment in the State 2 receptor population.

Effects of Monensin on Ethanol‐Induced Alterations in Function of Hepatocellular Asialoglycoprotein Receptor Subpopulations

Chronic ethanol consumption is associated with multiple impairments in receptor-mediated endocytosis (RME) in hepatocytes. RME mediated by the asialoglycoprotein receptor seems to be especially impaired by ethanol. In the present study, we determined susceptibility of RME to alterations in ethanol-fed and pair-fed control animals by the addition of a carboxylic ionophore, monensin. Monensin inhibits acidification of prelysosomal vesicular compartments, which results in a decrease in the rate of receptor-ligand dissociation within the cell. Low levels (25 microM) of monensin have been shown to preferentially affect receptor-ligand dissociation of one subset (state 2) of asialoglycoprotein receptor, whereas dissociation in a second subset (state 1 receptors) seems to be relatively unaffected. We examined whether ethanol treatment preferentially affected one or another of these receptor subpopulations. Male Wistar rats were fed a standard ethanol-containing (36% of calories) or control diet for 10 to 14 weeks, and hepatocytes were prepared from the animals. Similar to previous results from our laboratory, surface and total ligand and antibody binding were decreased by 30 to 45% (p < 0.01) in ethanol animals, compared with controls. An ethanol-induced impairment of receptor-ligand dissociation was also apparent in these cells, as shown by increased ratios of bound-to-free ligand during continuous endocytosis. After monensin treatment, surface receptors on both ethanol and control cells showed a similar pattern of redistribution to the cell interior and intracellular inactivation. When kinetics of intracellular receptor-ligand dissociation were examined upon addition of monensin, the bound-to-free ligand ratios in both control and ethanol cells increased dramatically and to an equal extent. These results indicate that, in the ethanol animals, the pattern of sensitivity to monensin is unchanged relative to controls. Thus, the relative proportion of state 1 and state 2 receptor populations do not seem to be affected after long-term feeding, and ethanol may be a perturbant that affects both state 1 and state 2 receptor function.

Mechanism of modulation of [ 3H]raclopride binding to dopaminergic receptors in rat striatal membranes by sodium ions

The mechanism of modulation of [ 3H]raclopride binding to dopaminergic receptors in rat brain striatal membranes by sodium ions was studied by means of equilibrium and kinetic measurements. Among different mono- and divalent cations studied, only sodium and lithium ions significantly enhanced [ 3H]raclopride binding to rat striatal membranes, but the effect of lithium was considerably smaller if compared with that of sodium. The equilibrium binding studies revealed that the increase in Na + concentration from 0.5 to 150 mM increased both the radioligand affinity and the number of binding sites. The meaning of these changes was established by kinetic studies, which yielded hyperbolic plots of [ 3H]raclopride binding rate constants over the radioligand concentration. These plots correspond to the two-step ligand binding reaction mechanism, involving fast binding equilibrium followed by a slow isomerization of the receptor-antagonist complex. Sodium ions did not influence the antagonist affinity for the receptor sites in the first step of the binding process, nor the rate of isomerization of the receptor-ligand complex, but slowed down the rate of deisomerization. This led to a change in the value of the receptor-ligand dissociation constant K d determined under equilibrium conditions. The same change in deisomerization rate was also sufficient to alter the receptor density ( B max), measured by the conventional ligand binding procedure.

Thermodynamic Parameters of Ligand-Receptor Interactions: Computation and Error Margins

A semiempirical relationship describing the temperature function of ligand-receptor dissociation constants (Kd), derived from heat capacities of the system in equilibrium, is suggested for computation of the standard enthalpy (δH°) and standard entropy (δS°) changes in equilibrium. The use of the linear expression (called Gibbs-van't Hoff equation) may lead to inaccurate results when heat capacity Cp displays a considerable temperature dependence. The accuracy of Kd, δH° and δS° has been studied by simulation experiments. In the case of Kd, deviations of computed from “true” values are determined by both the accuracy of experimental data used for its estimation, and by the shape of the binding isotherm (for instance, by Hill coefficients or by the presence of low affinity sites). As a rule, if errors of bound ligand measurements are greater than 20 per cent, Kd estimates ought to be considered as less reliable. However, computations of δH° and δS° that use such Kd values, are more correct, probably due to an error compensation. The suggested nonlinear temperature function of Kd enables an estimate of the heat capacity of the system and its temperature dependence.

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.

Fatty Acylation of the Rat Asialoglycoprotein Receptor: THE THREE SUBUNITS FROM ACTIVE RECEPTORS CONTAIN COVALENTLY BOUND PALMITATE AND STEARATE

Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomers composed of three homologous glycoprotein subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. ASGP-Rs mediate the endocytosis and degradation of circulating glycoconjugates containing terminal N-acetylgalactosamine or galactose, including desialylated plasma glycoproteins. We have shown in permeable rat hepatocytes that the ligand binding activity of one subpopulation of receptors (designated State 2 ASGP-Rs) can be decreased or increased, respectively, by ATP and palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). We proposed that a reversible and cyclic acylation/deacylation process may regulate ASGP-R activity during endocytosis, receptor-ligand dissociation, and receptor recycling. In the accompanying paper (Zeng, F-Y., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21388-21395), we show that the ligand binding activity of affinity-purified State 2 ASGP-Rs is decreased by treatment with hydroxylamine under mild conditions consistent with these ASGP-Rs being fatty acylated in vivo. In this study, we used a chemical method to determine the presence of covalently-bound fatty acids in individual ASGP-R subunits. The affinity-purified ASGP-R preparations were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, and the gel slices containing individual RHL subunits were treated with alkali to release covalently bound fatty acids, which were subsequently analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry. Both stearic and palmitic acids were detected in all three receptor subunits. Pretreatment of ASGP-Rs with hydroxylamine before SDS-polyacrylamide gel electrophoresis reduced the content of both fatty acids by 66-80%, indicating that most of these fatty acids are attached to cysteine residues via thioester linkages. Furthermore, when freshly isolated hepatocytes were cultured in the presence of [3H]palmitate, all three RHL subunits in affinity-purified ASGP-Rs were metabolically labeled. We conclude that RHL1, RHL2, and RHL3 are modified by fatty acylation in intact cells.

Evidence that the thyrotropin-releasing hormone receptor and its ligand are recycled dissociated from each other.

We have examined the trafficking of the thyrotropin-releasing hormone receptor (TRHR) and its ligand, after TRHR-TRH internalization in rat pituitary GH4C1 cells. After rapid ligand-induced receptor sequestration, the cell surface receptor pool was replenished. Replenishment was insensitive to inhibition of protein synthesis and was dependent on the duration of internalization; therefore, the replenished receptors were not newly synthesized but recycled. The total amount of recycled receptors decreased with increasing internalization time, resulting in only partial replenishment of the cell-surface receptor pool after prolonged incubation with ligand. Thus, in addition to a receptor recycling pathway, a non-cycling route exists for TRHR sorting; this route became dominant with increasing internalization periods. TRHR entry into these pathways was not determined by the affinity of the receptor-ligand interaction, because the extent of receptor recycling was similar after TRH- and methyl-TRH (MeTRH)-induced internalization. Unlike results with the TRHR, the TRH recycling pool was not depleted by the noncycling pathway. After multiple rounds of [3H]MeTRH internalization, the amount of cell-associated radioactivity increased with increasing internalization time due to accumulation of the ligand or its metabolites in a non-cycling pathway, but the absolute amount of recycled ligand remained constant after short or long internalization times. The difference in the proportion of TRHR and MeTRH that were diverted into a noncycling pathway indicated intracellular dissociation of the internalized TRHR-TRH complex. Dissociation of the internalized TRHR-TRH complex was dependent on the acidic pH in an intracellular compartment. Although extracellular acidic pH did not enhance cell-surface receptor-ligand (RL) dissociation, bafilomycin A1 inhibited both receptor and ligand recycling. We conclude that the TRHR-TRH system is unique among recycling receptors because, after RL sequestration, the TRHR-TRH complex becomes dissociated intracellularly via a bafilomycin A1-sensitive, acidic pH-dependent mechanism, and both the unoccupied TRHR and TRH recycle disassociated from each other.

Pituitary adenylate cyclase-activating polypeptide effects in pituitary cells: modulation by gonadotropin-releasing hormone in alpha T3-1 cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts via type I receptors in the pituitary to stimulate cAMP production. Gonadotropes are likely target cells for PACAP action, and we have recently shown alpha T3-1 cells, a clonal gonadotrope-derived cell line, to be PACAP responsive. Here we have explored the influence of GnRH on PACAP action in alpha T3-1 cells and show that PACAP38-stimulated cAMP production is inhibited by GnRH in both the presence and the absence of a phosphodiesterase inhibitor. This effect appears not to be Ca++ mediated but is mimicked by protein kinase C activation with phorbol 12-myristate 13-acetate. However, GnRH and phorbol 12-myristate 13-acetate do not inhibit binding of [125I]PACAP27 to intact alpha T3-1 cells, nor do they inhibit forskolin- or cholera toxin-stimulated cAMP accumulation, implying that the inhibitory effects are exerted at early stages in the PACAP receptor signaling pathway but distal to receptor occupancy. When cells were preincubated with PACAP38, extensive washing failed to prevent the stimulatory effect of the polypeptide presumably because of the slow rate of receptor-ligand dissociation. However, when the time course of PACAP38-stimulated effects on intracellular cAMP was assessed, the stimulatory effect of PACAP38 could be rapidly reversed by GnRH addition, and the inhibitory effect of GnRH was rapidly be reversed by a GnRH receptor antagonist. The data provide the first demonstration of cross-talk between phospholipase C and adenylate cyclase-activating peptides in gonadotrope-derived cells and establish the potential for hormonal modulation of PACAP action. We suggest that this inhibitory effect of GnRH might enable the releasing hormone to control the kinetics of cAMP signaling in gonadotropes in vivo.