Agonist-induced affinity alterations of a central nervous system alpha-bungarotoxin receptor

Abstract— The ability of cholinergic agonists to block the specific interaction of α-bungarotoxin (α-Bgt) with membrane-bound sites derived from rat brain is enhanced when membranes are preincubated with agonist. Thus, pretreatment of α-Bgt receptors with agonist (but not antagonist) causes transformation of sites to a high-affinity form toward agonist. This change in receptor state occurs with a half-time on the order of minutes, and is fully reversible on dilution of agonist. The results are consistent with the identity of α-Bgt binding sites as true central nicotinic acetylcholine receptors. Furthermore, this agonist-induced alteration in receptor state may represent an in vitro correlate of physiological desensitization. As determined from the effects of agonist on toxin binding isotherms, and on the rate of toxin binding to specific sites, agonist inhibition of toxin binding to the high-affinity state is non-competitive. This result suggests that there may exist discrete toxin-binding and agonist-binding sites on central toxin receptors.     

Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica

Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.     

Functional consequences of agonist-mediated state transitions in the cholinergic receptor. Studies in cultured muscle cells.

Parameters associated with activation and desensitization of the nicotinic receptor in the BC3H-1 muscle cell line have been compared with the state transitions that result upon combination with agonist. 125I-labeled cobra alpha-toxin is found to bind to an apparent single class of surface nicotinic receptors on the cells in situ with a rate constant of 1.15 x 10(5) M-1 s-1. The competition between cholinergic ligands and alpha-toxin reveals that agonists, but not classical antagonists, will promote a slow conversion to a receptor state where the affinity for agonists is enhanced. Moreover, agonists such as carbamylcholine elicit a permeability increase to 22Na+ ions that slowly decrements at a rate and to an extent closely paralleled by the conversion of the receptor to the high affinity state. Upon removal of the agonist, both the affinity increase and the diminished permeability change are completely reversible and again exhibit similar kinetics for their return to the original state. A comparison of the capacity of full agonists to compete with alpha-toxin binding and elicit a permeability change suggests that in the absence of agonist, receptor predominates in a low affinity activatable state. Binding of agonists to the low affinity state exhibits little if any cooperativity (n = 0.97 to 1.31), while the corresponding permeability change appears more cooperative (n = 1.31 to 1.52). By contrast, when receptors have been previously equilibrated with agonists, occupation of the receptor occurs over a 3- to 5-fold lower concentration range. Binding following equilibration closely correlates with a concomitant decrease in activatable receptor resulting from equivalent exposure to agonist. Furthermore, under equilibrium conditions, the binding of full agonists is typified by a moderate degree of homotropic cooperativity (1.25 to 1.44), enabling the receptor to desensitize over a narrow range of agonist concentration. Simultaneous measurement of occupation and activation parameters has enabled us to compare a state function for desensitization which is generated from binding parameters with the reduction in permeability seen in the desensitization process. A scheme describing the association of agonist with two functionally distinct receptor states is developed to account for the cooperative relationship between agonist binding and desensitization of the receptor.     

Functional consequences of the loss of high affinity agonist binding to gamma-aminobutyric acid type A receptors. Implications for receptor desensitization

We reported previously that tyrosine 62 of the beta2 subunit of the gamma-aminobutyric acid, type A (GABA(A)) receptor is an important determinant of high affinity agonist binding and that recombinant alpha1beta2gamma2(L) receptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50 approximately 0.7 microm, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the beta2 Y62S and Y62F mutations desensitized at slightly higher (2-7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABA(A) receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed.     

Differences between agonist and antagonist binding following beta-adrenergic receptor desensitization.

The specific beta-adrenergic agonist radioligand (+/-)-[3H]hydroxybenzylisoproterenol ([3H]HBI) was used to investigate alterations in the beta-adrenergic receptors of frog erythrocytes occurring during the process of agonist-induced, receptor-specific desensitization. There was close agreement between the percentage fall in [3H]HBI binding and that in catecholamine-stimulated adenylate cyclase activity following periods of preincubation of up to 7 h with 0.1 mM (-)-isoproterenol. Desensitization was maximal by 5 h, resulting in a 69% reduction in [3H]HBI binding and a 67% reduction in isoproterenol-stimulated adenylate cyclase activity. In contrast, binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol was significantly less affected by desensitization (p is less than 0.05 at 2 1/2, 5, and 7 h), showing a maximum reduction in binding of only 35% in these experiments. The consistent close agreement of reduction in agonist binding with that in hormone-stimulated adenylate cyclase activity, together with the significant difference observed between agonist and antagonist binding, implies that an alteration occurs during desensitization which preferentially interferes with agonist binding, while antagonist binding is less affected. The locus of this agonist-specific alteration may be the receptor binding site or a site involved in receptor-enzyme coupling. Agonist binding studies may now be used to assess more completely the desensitized state of beta-adrenergic receptors in systems in which marked desensitization of beta-adrenergic responses is associated with little or no reduction in antagonist binding.     

Activation and desensitization induce distinct conformational changes at the extracellular-transmembrane domain interface of the glycine receptor

Most ligand-gated channels exhibit desensitization, which is the progressive fading of ionic current in the prolonged presence of agonist. This process involves conformational changes that close the channel despite continued agonist binding. Despite the physiological and pathological importance of desensitization, little is known about the conformational changes that underlie this process in any Cys-loop ion channel receptor. Here we employed voltage clamp fluorometry to identify conformational changes that occur with a similar time course as the current desensitization rate in both slow- and fast-desensitizing α1 glycine receptor chloride channels. Voltage clamp fluorometry provides a direct indication of conformational changes that occur in the immediate vicinity of residues labeled with environmentally sensitive fluorophores. We compared the rates of current desensitization and fluorescence changes at nine labeled extracellular sites in both wild type slow-desensitizing and mutated (A248L) fast-desensitizing glycine receptors. As labels attached to three sites at the interface between the ligand binding domain and transmembrane domain reported fluorescence responses that changed in parallel with the current desensitization rate, we concluded that they experienced local conformational changes associated with desensitization. These labeled sites included A52C in loop 2, Q219C in the pre-M1 domain, and M227C in the M1 domain. Activation and desensitization were accompanied by physically distinct conformational changes at each labeled site. Because activation is mediated by a specific reorganization of molecular interactions at the extracellular-transmembrane domain interface, we propose that desensitization is mediated by a distinct set of conformational changes that prevents this reorganization from occurring, thereby favoring channel closure.     

Heterologous desensitization of adenylate cyclase with prostaglandin E1 alters sensitivity to inhibitory as well as stimulatory agonists

Adenylate cyclase in cultured human fibroblasts is activated by prostaglandin E1 (PGE1) or beta-adrenergic agonists, e.g., isoproterenol, and inhibited by muscarinic agonists. Incubation with PGE1 reduced adenylate cyclase responsiveness to both PGE1 and isoproterenol; this so-called heterologous desensitization is believed to result from impaired function of the stimulatory guanyl nucleotide-binding protein of the cyclase complex. The effect of heterologous desensitization by PGE1 on inhibition of adenylate cyclase by the muscarinic agonist oxotremorine was examined. Muscarinic inhibition of basal and isoproterenol-stimulated cAMP accumulation was attenuated following exposure to PGE1; the concentration of oxotremorine required for half-maximal inhibition of cAMP accumulation was increased. In both intact cells and membrane preparations the number of binding sites for [3H]scopolamine, a muscarinic antagonist, was unaltered by desensitization. Following exposure to PGE1, receptor affinity for oxotremorine, assessed by competition with [3H] scopolamine, and the guanyl nucleotide sensitivity of agonist binding were reduced. The amount of inhibitory guanyl nucleotide-binding regulatory protein available for [32P]ADP-ribosylation by pertussis toxin was unaltered by desensitization. Thus, heterologous desensitization of adenylate cyclase with the stimulatory agonist PGE1 alters sensitivity to inhibitory as well as stimulatory ligands.     

Site specificity of agonist-induced opening and desensitization of the Torpedo californica nicotinic acetylcholine receptor

Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to agonist-induced opening and desensitization. Timed dansyl-C6-choline (DC6C) binding followed by its dissociation upon mixing with high, competing agonist concentrations revealed four kinetic components: an initial, fast fluorescence decay, followed by a transient increase, and then two characteristic decays that reflect dissociation from the desensitized agonist sites. The transient increase resulted from DC6C binding to the open-channel based on its prevention by proadifen, a noncompetitive antagonist. Further characterization of DC6C channel binding by the inhibition of [3H]phencyclidine binding and by equilibrium measurements of DC6C fluorescence yielded KD values of 2-4 microM for the desensitized AChR and approximately 600 microM for the closed state. At this site, DC6C displayed a strongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer from tryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned to DC6C dissociation from the alphadelta site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and alpha-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 microM for the closed-state alphadelta site; the closed-state alphagamma-site affinity is inferred to be near 100 microM. These values and the known affinities for the desensitized conformation show that the alphagamma site drives AChR desensitization to a approximately 40-fold greater extent than the alphadelta site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.     

Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line

Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.     

Validation of Alexa-647-ATP as a powerful tool to study P2X receptor ligand binding and desensitization

Ion channel opening and desensitization is a fundamental process in neurotransmission. The ATP-gated P2X1 receptor (P2X1R) shows rapid and long-lasting desensitization upon agonist binding. This makes the electrophysiological investigation of its desensitization process, agonist unbinding, and recovery from desensitization a challenging task. Here, we show that the fluorescent agonist Alexa-647-ATP is a potent agonist at the P2X1R and a versatile tool to directly visualize agonist binding and unbinding. We demonstrate that the long-lasting desensitization of the P2X1R is due to both slow unbinding of agonist from the desensitized receptor and agonist mediated receptor internalization. Furthermore, the unbinding of the agonist Alexa-647-ATP from the desensitized receptor is accelerated in the continuous presence of competitive ligand. Modeling of our data indicates that three agonist molecules are required to drive the receptor into desensitization. Direct visualization of ligand unbinding from the desensitized receptor demonstrates the cooperativity of this process.     

Roles of agonist-binding sites in nicotinic acetylcholine receptor function

Under equilibrium conditions, the nicotinic acetylcholine receptor from Torpedo electroplax carries two high affinity-binding sites for agonists. It is generally assumed that these are the only agonist sites on the receptor and that their occupancy results in rapid channel activation followed by slower conformational transitions that lead to the high affinity equilibrium state. These slow transitions are thought to reflect the physiological process of desensitization. Here we show that preequilibration of the high affinity sites with saturating concentrations of carbamylcholine does not diminish the ion flux response to subsequent exposure to higher (activating) concentrations of this agonist. This finding has profound implications with respect to receptor function: (1) occupancy of the high affinity sites per se does not desensitize the receptor and (2) these sites cannot be directly involved in receptor activation. It is thus necessary to invoke the presence of additional binding sites in channel opening.     

Effects of Substance P on the Binding of Agonists to the Nicotinic Acetylcholine Receptor of Torpedo Electroplaque

Abstract: Abstract: The effect of the neuropeptide substance P on the binding of the cholinergic ligands to the nicotinic acetylcholine receptor of Torpedo electroplaque membranes was examined at a physiological concentration of NaCl (150 m M ). Substance P had no effect on the initial rate of ¹²⁵I-α-bungarotoxin binding at concentrations of <100 μ M . The peptide did not bind to the high-affinity local anesthetic site but allosterically modulated [³H]phencyclidine binding, positively in the absence of agonist and negatively in the presence of agonist. Substance P increased the apparent affinity of the cholinergic agonists carbamylcholine and acetylcholine at equilibrium. The effect of substance P on the equilibrium binding of [³H]acetylcholine was examined directly, and the peptide appeared to increase the affinity of the binding of the second molecule of agonist, with no effect on the binding of the first. This indicates that substance P can affect the cooperative interactions between agonist binding sites. Substance P appeared to increase the rate of carbamylcholine-induced desensitization; however, the data are also consistent with an allosteric mechanism that does not involve the desensitized state. To attempt to differentiate between these mechanisms, the rates of recovery were determined after exposure to peptide and/or agonist. The kinetics of recovery are consistent with stabilization of the desensitized state by substance P if the peptide remains bound long enough to allow rapid recovery to the low-affinity state. However, an allosteric modulation of agonist binding that does not involve the desensitized state cannot be ruled out.     

Agonist Regulation of Muscarinic Acetylcholine Receptors in Rat Spinal Cord

Abstract: In vitro studies with cultured cells originating from nervous tissue have shown that chronic exposure to muscarinic agonists results in a loss of muscarinic receptors. To determine whether this type of regulation of muscarinic receptor number also occurs in vivo , we infused carbachol into the spinal cords of rats. A single carbachol injection into the lumbar spinal cord caused a significant increase in the nociceptive threshold. This effect of carbachol diminished to control levels after 12 h of repeated agonist injections every 4 h and was blocked by atropine. The desensitization to the antinociceptive effects of carbachol was associated with a loss of muscarinic receptors as determined by the binding of the muscarinic antagonist [³H]quinuclidinyl benzilate. After a 24-h exposure to carbachol given every 4 h, there was about a 60% loss of binding sites. The loss of muscarinic receptors was also blocked by atropine and was reversible. These results represent direct evidence that a muscarinic agonist can regulate receptor number in the central nervous system and suggest that this loss of receptors is associated with a desensitization to the antinociceptive effects of carbachol injected into the spinal cord.     

The role of loop 5 in acetylcholine receptor channel gating

Nicotinic acetylcholine receptor channel (AChR) gating is an organized sequence of molecular motions that couples a change in the affinity for ligands at the two transmitter binding sites with a change in the ionic conductance of the pore. Loop 5 (L5) is a nine-residue segment (mouse alpha-subunit 92-100) that links the beta4 and beta5 strands of the extracellular domain and that (in the alpha-subunit) contains binding segment A. Based on the structure of the acetylcholine binding protein, we speculate that in AChRs L5 projects from the transmitter binding site toward the membrane along a subunit interface. We used single-channel kinetics to quantify the effects of mutations to alphaD97 and other L5 residues with respect to agonist binding (to both open and closed AChRs), channel gating (for both unliganded and fully-liganded AChRs), and desensitization. Most alphaD97 mutations increase gating (up to 168-fold) but have little or no effect on ligand binding or desensitization. Rate-equilibrium free energy relationship analysis indicates that alphaD97 moves early in the gating reaction, in synchrony with the movement of the transmitter binding site (Phi = 0.93, which implies an open-like character at the transition state). alphaD97 mutations in the two alpha-subunits have unequal energetic consequences for gating, but their contributions are independent. We conclude that the key, underlying functional consequence of alphaD97 perturbations is to increase the unliganded gating equilibrium constant. L5 emerges as an important and early link in the AChR gating reaction which, in the absence of agonist, serves to increase the relative stability of the closed conformation of the protein.     

Measurement of conformational changes accompanying desensitization in an ionotropic glutamate receptor

The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.     

Activation and desensitization of the recombinant P2X1 receptor at nanomolar ATP concentrations

Activation and desensitization kinetics of the rat P2X1 receptor at nanomolar ATP concentrations were studied in Xenopus oocytes using two-electrode voltage-clamp recording. The solution exchange system used allowed complete and reproducible solution exchange in <0.5 s. Sustained exposure to 1-100 nM ATP led to a profound desensitization of P2X1 receptors. At steady-state, desensitization could be described by the Hill equation with a K1/2 value of 3.2 +/- 0.1 nM. Also, the ATP dependence of peak currents could be described by a Hill equation with an EC50 value of 0.7 microM. Accordingly, ATP dose-effect relationships of activation and desensitization practically do not overlap. Recovery from desensitization could be described by a monoexponential function with the time-constant tau = 11.6 +/-1.0 min. Current transients at 10-100 nM ATP, which elicited 0.1-8.5% of the maximum response, were compatible with a linear three-state model, C-O-D (closed-open-desensitized), with an ATP concentration-dependent activation rate and an ATP concentration-independent (constant) desensitization rate. In the range of 18-300 nM ATP, the total areas under the elicited current transients were equal, suggesting that P2X1 receptor desensitization occurs exclusively via the open conformation. Hence, our results are compatible with a model, according to which P2X1 receptor activation and desensitization follow the same reaction pathway, i.e., without significant C to D transition. We assume that the K1/2 of 3.2 nM for receptor desensitization reflects the nanomolar ATP affinity of the receptor found by others in agonist binding experiments. The high EC50 value of 0.7 microM for receptor activation is a consequence of fast desensitization combined with nonsteady-state conditions during recording of peak currents, which are the basis of the dose-response curve. Our results imply that nanomolar extracellular ATP concentrations can obscure P2X1 receptor responses by driving a significant fraction of the receptor pool into a long-lasting refractory closed state.     

Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the G protein-coupled receptor kinase site serine cluster in beta2-adrenergic receptor internalization, desensitization, and beta-arrestin translocation

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.     

Subtype-selective desensitization of alpha 2-adrenergic receptors. Different mechanisms control short and long term agonist-promoted desensitization of alpha 2C10, alpha 2C4, and alpha 2C2.

We have recently shown that the alpha 2C10 adrenergic receptor (AR) undergoes short term agonist-promoted desensitization, mediated by phosphorylation of sites in the third intracellular loop. There is significant divergence in the third loop amino acid sequences between alpha 2C10 and the other subtypes, alpha 2C4 and alpha 2C2. We therefore explored the mechanisms of alpha 2AR subtype desensitization by expressing each human subtype in Chinese hamster ovary cells and subjecting them to short and long term epinephrine exposures. After 30 min of agonist exposure, alpha 2C10 and alpha 2C2 displayed desensitization characterized by rightward shifts in the curves for epinephrine-mediated inhibition of adenylyl cyclase (EC50 = alpha 2C10, 0.24 +/- 0.02 microM increasing to 1.1 +/- 0.1 microM; alpha 2C2, 1.3 +/- 0.3 increasing to 2.6 +/- 0.3 microM). Coincident with alpha 2C10 and alpha 2C2 desensitizations were decreases in agonist high affinity binding. In contrast, alpha 2C4 underwent no functional desensitization after short term agonist exposure, nor were there any changes in agonist high affinity binding. Agonist-promoted receptor sequestration was clearly greater with alpha 2C10 (approximately 26%) and alpha 2C2 (approximately 35%) as compared to alpha 2C4 (approximately 12%), but such sequestration did not play a significant role in short term desensitization, as blockade with concanavalin A had no effect on desensitization patterns. In contrast to these findings, all alpha 2AR subtypes underwent desensitization after prolonged (24 h) agonist exposure. However, alpha 2C10 and alpha 2C2 displayed substantially more desensitization (approximately 20-fold increase in EC50) as compared to alpha 2C4 (approximately 5-fold increase). The primary mechanism of desensitization during long term agonist exposure was found to be a decrease in the amount of cellular Gi, which was equivalent in magnitude in cells expressing all three subtypes. However, in addition to a decrease in Gi, alpha 2C10 and alpha 2C2 underwent down-regulation of receptor levels during long term agonist exposure, while alpha 2C4 did not. Given that all three subtypes bind endogenous catecholamines with high affinity and inhibit adenylyl cyclase efficiently, the significance of multiple subtypes has heretofore been obscure. Our results show that alpha 2AR undergo subtype-selective desensitization to agonists and suggest that alpha 2AR subtypes may have evolved to meet differing needs for adaptive regulation.     

Regulation of Muscarinic Acetylcholine Receptor Concentration in Cloned Neuroblastoma Cells

Abstract: The concentration of muscarinic acetylcholine receptors in the neuroblastoma cell line NIE-115 is regulated by receptor activation. Muscarinic agonists cause a time and dose-dependent loss of [³H]quinuclidinyl benzilate binding sites from cultured cells. Muscarinic antagonists have no effect on receptor concentration and block agonist-induced regulation. The maximum decrease in steady state receptor levels is 80% and occurs within 9 h. The altered steady state concentration persists as long as agonist remains present. Upon withdrawal of agonist, the concentration of receptors returns to control levels. This increase requires protein synthesis. Kinetically, the increase in receptors following withdrawal of agonist is slower than the decrease caused by addition of agonist, suggesting that bursts of receptor activation could lower receptor levels. In harmony with this prediction, cycles in which receptors are active for 15 min and then inactive for 15 min cause a 50% decrease in receptor concentration in a 6-h period.