The differential expression of the $\beta\sb2$-adrenergic receptor modifies agonist-dependent adenylylcyclase activation

There have been multiple reports which indicate that variations in $\beta$AR expression affect the V$\sb{\rm max}$ observed for the agonist-dependent activation of adenylylcyclase. This observation has been ignored by most researchers when V$\sb{\rm max}$ values obtained for wild type and mutant receptors are compared. Such an imprecise analysis may lead to erroneous conclusions concerning the ability of a receptor to activate adenylylcyclase. Equations were derived from the Cassel-Selinger model of GTPase activity and Tolkovsky and Levitzki's Collision Coupling model which predict that the EC$\sb{50}$ and V$\sb{\rm max}$ for the activation of adenylylcyclase are a function of receptor number. Experimental results for L cell clones in which either hamster or human $\beta$AR were transfected at varying levels showed that EC$\sb{50}$ decreases and V$\sb{\rm max}$ increases as receptor number increases. Comparison of these results with simulations obtained from the equations describing EC$\sb{50}$ and V$\sb{\rm max}$ showed a close correlation. This documents that the kinetic parameters of adenylylcyclase activation change with the level of receptor expression and relates this phenomenon to a theoretical framework concerning the mechanisms involved in $\beta$AR signal transduction.^ One of the terms used in the equations which expressed the EC$\sb{50}$ and V$\sb{\rm max}$ as a function of receptor number is coupling efficiency, defined as $\rm k\sb1/k\sb{-1}$. Calculation of $\rm k\sb1/k\sb{-1}$ can be accomplished for wild type receptors with the easily measured experimental values of agonist K$\sb{\rm d}$, EC$\sb{50}$ and receptor number. This was demonstrated for hamster $\beta$AR which yielded a coupling efficiency of 0.15 $\pm$ 0.003 and human $\beta$AR which yielded a coupling efficiency of 0.90 $\pm$ 0.031. $\rm k\sb1/k\sb{-1}$ replaces the traditional qualitative evaluation of the ability to activate adenylylcyclase, which utilizes V$\sb{\rm max}$ without correction for variation in receptor number, with a quantitative definition that more accurately describes the ability of $\beta$AR to couple to G$\sb{\rm s}$.^ The equations which express the EC$\sb{50}$ and V$\sb{\rm max}$ for adenylylcyclase activation as a function of receptor number and coupling efficiency were tested to determine whether they could accurately simulate the changes seen in these parameters during desensitization. Data from original desensitization experiments and data from the literature (24,25,52,54,83) were compared to simulated changes in EC$\sb{50}$ and V$\sb{\rm max}$. In a variety of systems the predictions of the equations were consistent with the changes observed in EC$\sb{50}$ and V$\sb{\rm max}$. In addition reductions in the calculated value of $\rm k\sb1/k\sb{-1}$ was shown to correlate well with $\beta$AR phosphorylation and to be minimally affected by sequestration and down-regulation. ^

Effects of variable Gs expression on $\beta\sb2$-adrenergic receptor stimulated adenylyl cyclase response

An exact knowledge of the kinetic nature of the interaction between the stimulatory G protein (G$\sb{\rm s}$) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of Gs mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. In particular, insight as to the association of these proteins could lead to progress in tumor biology where single spontaneous mutations in G proteins have been associated with the formation of tumors (118). The question this work attempts to answer is whether the adenylyl cyclase activation by epinephrine stimulated $\beta\sb2$-adrenergic receptors occurs via G$\sb{\rm s}$ proteins by a G$\sb{\rm s}$ to C shuttle or G$\sb{\rm s}$-C precoupled mechanism. The two forms of activation are distinguishable by the effect of G$\sb{\rm s}$ levels on epinephrine stimulated EC50 values for cyclase activation.^ We have made stable transfectants of S49 cyc$\sp-$ cells with the gene for the $\alpha$ protein of G$\sb{\rm s}$ $(\alpha\sb{\rm s})$ which is under the control of the mouse mammary tumor virus LTR promoter (110). Expression of G$\sb{\rm s}\alpha$ was then controlled by incubation of the cells for various times with 5 $\mu$M dexamethasone. Expression of G$\sb{\rm s}\alpha$ led to the appearance of GTP shifts in the competitive binding of epinephrine with $\sp{125}$ICYP to the $\beta$-adrenergic receptors and to agonist dependent adenylyl cyclase activity. High expression of G$\sb{\rm s}\alpha$ resulted in lower EC50's for the adenylyl cyclase activity in response to epinephrine than did low expression. By kinetic modelling, this result is consistent with the existence of a shuttle mechanism for adenylyl cyclase activation by hormones.^ One item of concern that remains to be addressed is the extent to which activation of adenylyl cyclase occurs by a "pure" shuttle mechanism. Kinetic and biochemical experiments by other investigators have revealed that adenylyl cyclase activation, by hormones, may occur via a Gs-C precoupled mechanism (80, 94, 97). Activation of adenylyl cyclase, therefore, probably does not occur by either a pure "'Shuttle" or "Gs-C Precoupled" mechanism, but rather by a "Hybrid" mechanism. The extent to which either the shuttle or precoupled mechanism contributes to hormone stimulated adenylyl cyclase activity is the subject of on-going research. ^

$\beta\sb2$-adrenergic receptor desensitization, internalization and phosphorylation in response to full and partial agonists

The objective of this study is to test the hypothesis that partial agonists produce less desensitization because they generate less of the active conformation of the $\beta\sb2$-adrenergic receptor ($\beta$AR) (R*) and in turn cause less $\beta$AR phosphorylation by beta adrenergic receptor kinase ($\beta$ARK) and less $\beta$AR internalization. In the present work, rates of desensitization, internalization, and phosphorylation caused by a series of $\beta$AR agonists were correlated with a quantitative measure, defined as coupling efficiency, of agonist-dependent $\beta$AR activation of adenylyl cyclase. These studies were preformed in HEK-293 cells overexpressing the $\beta$AR with hemagglutinin (HA) and 6-histidine (6HIS) epitopes introduced into the N- and C-termini respectively. Agonists chosen provided a 95-fold range of coupling efficiencies, and, relative to epinephrine, the best agonist, (100%) were fenoterol (42%), albuterol (4.9%), dobutamine (2.5%) and ephedrine (1.1%). At concentrations of these agonists yielding $>$90% receptor occupancy, the rate and extent of the rapid phase (0-30 min) of agonist induced desensitization of adenylyl cyclase followed the same order as coupling efficiency, that is, epinephrine $\ge$ fitnoterol $>$ albuterol $>$ dobutamine $>$ ephedrine. The rate of internalization, measured by a loss of surface receptors during desensitization, with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like desensitization and internalization, $\beta$AR phosphorylation exhibited a dependency on agonist strength. The two strongest agonists epinephrine and fenoterol provoked 11 to 13 fold increases in the level of $\beta$AR phosphorylation after just 1 min, whereas the weakest agonists dobutamine and ephedrine caused only 3 to 4 fold increases in phosphorylation. With longer treatment times, the level of $\beta$AR phosphorylation declined with the strong agonists, but progressively increased with the weaker partial agonists. The major conclusion drawn from this study is that the occupancy-dependent rate of receptor phosphorylation increases with agonist coupling efficiencies and that this is sufficient to explain the desensitization, internalization, and phosphorylation data obtained.^ The mechanism of activation and desensitization by the partial $\beta$AR agonist salmeterol was also examined in this study. This drug is extremely hydrophobic and its study presents possibly unique problems. To determine whether salmeterol induces desensitization of the $\beta$AR its action has been studied using our system. Employing the use of reversible antagonists it was found that salmeterol, which has an estimated coupling efficiency near that of albuterol caused $\beta$AR desensitization. This desensitization was much reduced relative to epinephrine. Consistent with its coupling efficiency, it was found to be similar to albuterol in its ability to induce internalization and phosphorylation of the $\beta$AR. (Abstract shortened by UMI.) ^

Evidence for the role of agonist binding frequency in receptor -mediated activation of adenylate cyclase

$\beta$-adrenergic receptor-mediated activation of adenylate cyclase exhibits an agonist-specific separation between the dose/response curve (characterized by the EC$\sb{50}$) and the dose/binding curve (characterized by the K$\sb{\rm d}$). Cyclase activity can be near-maximal when receptor occupancy is quite low (EC$\sb{50}$ $\ll$ K$\sb{\rm d}$). This separation between the binding and response curves can be explained by the assumption that the rate of cyclase activation is proportional to the concentration of agonist-bound receptors, since the receptor is mobile and can activate more than one cyclase (the Collision Coupling Model of Tolkovsky and Levitzki). Here it is established that agonist binding frequency plays an additional role in adenylate cyclase activation in S49 murine lymphoma cells. Using epinephrine (EC$\sb{50}$ = 10 nM, K$\sb{\rm d}$ = 2 $\mu$M), the rate of cyclase activation decreased by 80% when a small (1.5%) receptor occupancy was restricted (by addition of the antagonist propranolol) to a small number (1.5%) of receptors rather than being proportionally distributed among the cell's entire population of receptors. Thus adenylate cyclase activity is not proportional to receptor occupancy in all circumstances. Collisions between receptor and cyclase pairs apparently occur a number of times in rapid sequence (an encounter); the high binding frequency of epinephrine ensures that discontiguous regions of the cell surface experience some period of agonist-bound receptor activity per small unit time minimizing "wasted" collisions between activated cyclase and bound receptor within an encounter. A contribution of agonist binding frequency to activation is thus possible when: (1) the mean lifetime of the agonist-receptor complex is shorter than the mean encounter time, and (2) the absolute efficiency (intrinsic ability to promote cyclase activation per collision) of the agonist-receptor complex is high. These conclusions are supported by experiments using agonists of different efficiencies and binding frequencies. These results are formalized in the Encounter Coupling Model of adenylate cyclase activation, which takes into explicit account the agonist binding frequency, agonist affinity for the $\beta$-adrenergic receptor, agonist efficiency, encounter frequency and the encounter time between receptor and cyclase. ^

The two -state model of receptor activation: The agonist and the efficacy

The Two State model describes how drugs activate receptors by inducing or supporting a conformational change in the receptor from “off” to “on”. The beta 2 adrenergic receptor system is the model system which was used to formalize the concept of two states, and the mechanism of hormone agonist stimulation of this receptor is similar to ligand activation of other seven transmembrane receptors. Hormone binding to beta 2 adrenergic receptors stimulates the intracellular production of cyclic adenosine monophosphate (cAMP), which is mediated through the stimulatory guanyl nucleotide binding protein (Gs) interacting with the membrane bound enzyme adenylylcyclase (AC). ^ The effects of cAMP include protein phosphorylation, metabolic regulation and transcriptional regulation. The beta 2 adrenergic receptor system is the most well known of its family of G protein coupled receptors. Ligands have been scrutinized extensively in search of more effective therapeutic agents at this receptor as well as for insight into the biochemical mechanism of receptor activation. Hormone binding to receptor is thought to induce a conformational change in the receptor that increases its affinity for inactive Gs, catalyzes the release of GDP and subsequent binding of GTP and activation of Gs. ^ However, some beta 2 ligands are more efficient at this transformation than others, and the underlying mechanism for this drug specificity is not fully understood. The central problem in pharmacology is the characterization of drugs in their effect on physiological systems, and consequently, the search for a rational scale of drug effectiveness has been the effort of many investigators, which continues to the present time as models are proposed, tested and modified. ^ The major results of this thesis show that for many b2 -adrenergic ligands, the Two State model is quite adequate to explain their activity, but dobutamine (+/−3,4-dihydroxy-N-[3-(4-hydroxyphenyl)-1-methylpropyl]- b -phenethylamine) fails to conform to the predictions of the Two State model. It is a weak partial agonist, but it forms a large amount of high affinity complexes, and these complexes are formed at low concentrations much better than at higher concentrations. Finally, dobutamine causes the beta 2 adrenergic receptor to form high affinity complexes at a much faster rate than can be accounted for by its low efficiency activating AC. Because the Two State model fails to predict the activity of dobutamine in three different ways, it has been disproven in its strictest form. ^

Reversal of myocyte hypertrophy by ventricular unloading: cardiac improvement without adrenergic receptor up-regulation and relocalization.

In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement. We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.

Opioid dependence treatment: options in pharmacotherapy.

The development of effective treatments for opioid dependence is of great importance given the devastating consequences of the disease. Pharmacotherapies for opioid addiction include opioid agonists, partial agonists, opioid antagonists, and alpha-2-adrenergic agonists, which are targeted toward either detoxification or long-term agonist maintenance. Agonist maintenance therapy is currently the recommended treatment for opioid dependence due to its superior outcomes relative to detoxification. Detoxification protocols have limited long-term efficacy, and patient discomfort remains a significant therapy challenge. Buprenorphine's effectiveness relative to methadone remains a controversy and may be most appropriate for patients in need of low doses of agonist treatment. Buprenorphine appears superior to alpha-2 agonists, however, and office-based treatment with buprenorphine in the USA is gaining support. Studies of sustained-release formulations of naltrexone suggest improved effectiveness for retention and sustained abstinence; however, randomized clinical trials are needed.

MODELLING β2AR REGULATION

The β2 adrenergic receptor (β2AR) regulates smooth muscle relaxation in the vasculature and airways. Long- and Short-acting β-agonists (LABAs/SABAs) are widely used in treatment of chronic obstructive pulmonary disorder (COPD) and asthma. Despite their widespread clinical use we do not understand well the dominant β2AR regulatory pathways that are stimulated during therapy and bring about tachyphylaxis, which is the loss of drug effects. Thus, an understanding of how the β2AR responds to various β-agonists is crucial to their rational use. Towards that end we have developed deterministic models that explore the mechanism of drug- induced β2AR regulation. These mathematical models can be classified into three classes; (i) Six quantitative models of SABA-induced G protein coupled receptor kinase (GRK)-mediated β2AR regulation; (ii) Three phenomenological models of salmeterol (a LABA)-induced GRK-mediated β2AR regulation; and (iii) One semi-quantitative, unified model of SABA-induced GRK-, protein kinase A (PKA)-, and phosphodiesterase (PDE)-mediated regulation of β2AR signalling. The various models were constrained with all or some of the following experimental data; (i) GRK-mediated β2AR phosphorylation in response to various LABAs/SABAs; (ii) dephosphorylation of the GRK site on the β2AR; (iii) β2AR internalisation; (iv) β2AR recycling; (v) β2AR desensitisation; (vi) β2AR resensitisation; (vii) PKA-mediated β2AR phosphorylation in response to a SABA; and (viii) LABA/SABA induced cAMP profile ± PDE inhibitors. The models of GRK-mediated β2AR regulation show that plasma membrane dephosphorylation and recycling of the phosphorylated β2AR are required to reconcile with the measured dephosphorylation kinetics. We further used a consensus model to predict the consequences of rapid pulsatile agonist stimulation and found that although resensitisation was rapid, the β2AR system retained the memory of prior stimuli and desensitised much more rapidly and strongly in response to subsequent stimuli. This could explain tachyphylaxis of SABAs over repeated use in rescue therapy of asthma patients. The LABA models show that the long action of salmeterol can be explained due to decreased stability of the arrestin/β2AR/salmeterol complex. This could explain long action of β-agonists used in maintenance therapy of asthma patients. Our consensus model of PKA/PDE/GRK-mediated β2AR regulation is being used to identify the dominant β2AR desensitisation pathways under different therapeutic regimens in human airway cells. In summary our models represent a significant advance towards understanding agonist-specific β2AR regulation that will aid in a more rational use of the β2AR agonists in the treatment of asthma.

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^

Regulation of adenylyl cyclase by protein kinase C and P$\sb2$ purinergic agonists

Activation of protein kinase C (PKC) causes multiple effects on adenylyl cyclase (AC), (i) an inhibition of (hormone) receptor/G$\sb{\rm s}$ coupling, consistent with PKC modification of the receptor and (ii) a postreceptor sensitization consistent with a PKC-mediated modification of the stimulatory (G$\sb{\rm s}$) or inhibitory (G$\sb{\rm i}$) G-proteins or the catalyst (C) of AC. In L cells expressing the wild-type beta-adrenergic receptor ($\beta$AR) 4-$\beta$ phorbol 12-myristate-13-acetate (PMA) caused 2-3-fold increases in the K$\sb{\rm act}$ and V$\sb{\rm max}$ for epinephrine-stimulated AC activity and an attenuation of GTP-mediated inhibition of AC. Deletion of a concensus site for PKC phosphorylation (amino acids 259-262) from the $\beta$AR eliminated the PMA-induced increase in the K$\sb{\rm act}$, but had no effect on the other actions of PMA. PMA also increased the K$\sb{\rm act}$ and V$\sb{\rm max}$ for prostaglandin E$\sb1$ (PGE$\sb1$)-stimulated AC and the V$\sb{\rm max}$ for forskolin-stimulated AC. Maximal PMA-induced sensitizations were observed when AC was assayed in the presence of 10 $\mu$M GTP and 0.3 mM (Mg$\sp{++}$).^ Liao et al. (J. Biol. Chem. 265:11273-11284 (1990)) have shown that the P$\sb2$ purinergic receptor agonist ATP stimulates hydrolysis of 4,5 inositol bisphosphate (PIP$\sb2$) by phospholipase C (PLC) in L cells. To determine if agonists that stimulate PLC and PMA had similar effects on AC function we compared the effects of ATP and PMA. ATP caused a rapid 50-150% sensitization of PGE$\sb1$-, epinephrine-, and forskolin-stimulated AC activity with an EC$\sb{50}$ of 3 $\mu$M ATP. The sensitization was similar (i.e. Mg$\sp{++}$ and GTP sensitivity) to that caused by 10 nM PMA. However, unlike PMA ATP did not affect the K$\sb{\rm act}$ for hormone-stimulated AC and its effects were unaltered by down-regulation of PKCs following long term PMA treatment. Our results demonstrate that a PKC concensus site in the $\beta$AR, is required for the PMA-induced decrease in receptor/G$\sb{\rm s}$ coupling. Our data also indicate that activation of P$\sb2$ purinergic receptors by ATP may be important in the sensitization of AC in L cells. The mechanism behind this effect remains to be determined. ^

Receptor-mediated adenylylcyclase activation following ``homologous'' and ``heterologous'' desensitization

Using a "collision-coupling" model for $\beta \sb 2$-adrenergic receptor-mediated activation of adenylylcyclase in S49 lymphoma cells, the rate-limiting step of that activation was identified as the association of an "active-state", hormone-bound receptor (HR$\sp\*$) with a G$\sb{\rm s}$-adenylylcyclase moiety (G$\sb{\rm s}$C). It was subsequently hypothesized that the location of the rate-limiting step would not be shifted elsewhere in the activation scheme by receptor desensitization. The traditional focus of receptor desensitization studies has been on modifications of the receptor molecule itself. A "clear-cut" answer to the present hypothesis provides new information on modifications in the function of the receptor following desensitization.^ "Heterologous" desensitization was induced in wild type S49 cells with agents which increase intracellular cAMP without occupying $\beta\sb2$-adrenergic receptors; PGE$\sb1$, forskolin and dibutyryl cAMP. These treatments avoided overlapping effects on $\beta\sb2$-adrenergic receptors by the "homologous" mechanism, in which occupancy by hormone is causative. Although the steady-state activation rate was decreased following heterologous desensitization, that rate was still limited by the association between HR* and G$\sb{\rm s}$C. Thus "heterologous" desensitization acts at the equilibrium between HR and HR* (which is driven by hormone efficiency) such that HR* formation becomes less likely and the frequency of HR*G$\sb{\rm s}$C associations decreases.^ "Homologous" desensitization was induced by high (1-10$\mu$M) epinephrine concentrations in the S49 variant deficient in cAMP-dependent protein kinase, KIN$\sp-$. Use of KIN$\sp-$minimized overlapping effects by the "heterologous" mechanism, which is PKA-dependent. Following homologous desensitization, roughly 50% of the receptors in plasma membrane preparations no longer formed HR*G$\sb{\rm s}$C complexes; evidenced by a decrease in high-affinity hormone binding sites. The loss of HR*G$\sb{\rm s}$C formation did not appear related to the HR/HR* equilibrium. Increasing the efficiency of the assay agonist did nothing to "override" the effect. HR*G$\sb{\rm s}$C association was still the rate-limiting step among the remaining functional receptors. It was not distinguishable whether the remaining activity was "desensitized" due to adenylylcyclase having decreased access to receptors within plasma membrane fragments or due to an effect similar to "heterologous" desensitization. ^

Molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$-adrenergic receptor

The $\beta$-adrenergic receptor ($\beta$AR), which couples to G$\sb{\rm s}$ and activates adenylylcyclase, has been a prototype for studying the activation and desensitization of G-protein-coupled receptors. The main objective of the present study is to elucidate the molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$AR.^ Activation of cAPK or PKC causes a rapid desensitization of $\beta$AR stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the $\beta$AR, RRSSK$\sp{263}$. To determine the role of the individual serines in the cAPK- and PKC-meditated desensitizations, wild type (WT) and mutant $\beta$ARs containing the substitutions, Ser$\sp{261} \to$ A, Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and Ser$\sp{261/262} \to$ A, were constructed and stably transfected into L cells. The cAPK-mediated desensitization was decreased 70-80% by the Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and the Ser$\sp{261/262} \to$ A mutations, but was not altered by the Ser$\sp{261} \to$ A substitution, demonstrating that Ser$\sp{262}$ was the primary site of the cAPK-induced desensitization. The PMA/PKC-induced desensitization was unaffected by either of the single serine to alanine substitutions, but was reduced 80% by the double serine to alanine substitution, suggesting that either serine was sufficient to confer the PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser$\sp{262} \to$ A, was partially uncoupled. The Ser$\sp{262} \to$ D mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor.^ To accomplish the in vivo phosphorylation of the $\beta$AR, we used two epitope-modified $\beta$ARs, hemagglutinin-tagged $\beta$AR (HA-$\beta$AR) and 6 histidine-tagged $\beta$AR (6His-$\beta$AR), for a high efficiency purification of the $\beta$AR. Neither HA-$\beta$AR nor 6His-$\beta$AR altered activation and desensitization of the $\beta$AR significantly as compared to unmodified wild type $\beta$AR. 61% recovery of ICYP-labeled $\beta$AR was obtained with Ni-NTA column chromatography.^ The truncation 354 mutant $\beta$AR(T354), lacking putative $\beta$ARK site(s), displayed a normal epinephrine stimulation of adenylylcyclase. Although 1.0 $\mu$M epinephrine induced 60% less desensitization in T354 as compared to wild type $\beta$AR, 1.0 $\mu$M epinephrine-mediated desensitization in T354 was 35% greater than PGE$\sb1$-mediated desensitization, which is essentially identical in both WT and T354. These results suggested that sequences downstream of residue 354 may play a role in homologous desensitization and that internalization may be attributed to the additional desensitization besides the cAMP mechanism in T354 $\beta$AR. (Abstract shortened by UMI.) ^

TIME DEPENDENT ALTERATIONS IN CENTRAL NERVOUS SYSTEM DOPAMINE RECEPTORS INDUCED BY DOPAMINE AGONIST TREATMENT

Levodopa, the precursor of dopamine, is currently the drug of choice in the treatment of Parkinson's disease. Recently, two direct dopamine agonists, bromocriptine and pergolide, have been tested for the treatment of Parkinson's disease because of reduced side effects compared to levodopa. Few studies have evaluated the effects of long-term treatment of dopamine agonists on dopamine receptor regulation in the central nervous system. Thus, the purpose of this study was to determine whether chronic dopamine agonist treatment produces a down-regulation of striatal dopamine receptor function and to compare the results of the two classes of dopaminergic drugs.^ Levodopa with carbidopa, a peripheral decarboxylase inhibitor, was administered orally to rats whereas bromocriptine and pergolide were injected intraperitoneally once daily. Several neurochemical parameters were examined from 1 to 28 days.^ Levodopa minimally decreased striatal D-1 receptor activity but increased the number of striatal D-2 binding sites. Levodopa increased the V(,max) of tyrosine hydroxylase (TH) in all brain regions tested. Protein blot analysis of striatal TH indicated a significant increase in the amount of TH present. Dopamine-beta-hydroxylase (DBH) activity was markedly decreased in all brain regions studied and mixing experiments of control and drug-treated cortices did not show the presence of an increased level of endogenous inhibitors.^ Bromocriptine treatment decreased the number of D-2 binding sites. Striatal TH activity was decreased and protein blot analysis indicated no change in TH quantity. The specificity of bromocriptine for striatal TH suggested that bromocriptine preferentially interacts with dopamine autoreceptors.^ Combination levodopa-bromocriptine was administered for 12 days. There was a decrease in both D-1 receptor activity and D-2 binding sites, and a decrease in brain HVA levels suggesting a postsynaptic receptor action. Pergolide produced identical results to the combination levodopa-bromocriptine studies.^ In conclusion, combination levodopa-bromocriptine and pergolide treatments exhibited the expected down-regulation of dopamine receptor activity. In contrast, levodopa appeared to up-regulate dopamine receptor activity. Thus, these data may help to explain, on a biochemical basis, the decrease in the levodopa-induced side effects noted with combination levodopa-bromocriptine or pergolide therapies in the treatment of Parkinson's disease. ^

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

Identification of a conserved cluster in the RH domain of GRK critical for activation by GPCRs

One of the most critical aspects of G Protein Coupled Receptors (GPCRs) regulation is their rapid and acute desensitization following agonist stimulation. Phosphorylation of these receptors by GPCR kinases (GRK) is a major mechanism of desensitization. Considerable evidence from studies of rhodopsin kinase and GRK2 suggests there is an allosteric docking site for the receptor distinct from the GRK catalytic site. While the agonist-activated GPCR appears crucial for GRK activation, the molecular details of this interaction remain unclear. Recent studies suggested an important role for the N- and C-termini and domains in the small lobe of the kinase domain in allosteric activation; however, neither the mechanism of action of that site nor the RH domain contributions have been elucidated. To search for the allosteric site, we first indentified evolutionarily conserved sites within the RH and kinase domains presumably deterministic of protein function employing evolutionary trace (ET) methodology and crystal structures of GRK6. Focusing on a conserved cluster centered on helices 3, 9, and 10 in the RH domain, key residues of GRK5 and 6 were targeted for mutagenesis and functional assays. We found that a number of double mutations within helices 3, 9, and 10 and the N-terminus markedly reduced (50–90%) the constitutive phosphorylation of the β-2 Adrenergic Receptor (β2AR) in intact cells and phosphorylation of light-activated rhodopsin (Rho*) in vitro as compared to wild type (WT) GRK5 or 6. Based on these results, we designed peptide mimetics of GRK5 helix 9 both computationally and through chemical modifications with the goal of both confirming the importance of helix 9 and developing a useful inhibitor to disrupt the GPCR-GRK interaction. Several peptides were found to block Rho* phosphorylation by GRK5 including the native helix 9 sequence, Peptide Builder designed-peptide preserving only the key ET residues, and chemically locked helices. Most peptidomimetics showed inhibition of GRK5 activity greater than 80 % with an IC50 of ∼ 30 µM. Alanine scanning of helix 9 has further revealed both essential and non-essential residues for inhibition. Importantly, substitution of Arg 169 by an alanine in the native helix 9-based peptide gave an almost complete inhibition at 30 µM with an IC50 of ∼ 10 µM. In summary we report a previously unrecognized crucial role for the RH domain of GRK5 and 6, and the subsequent identification of a lead peptide inhibitor of protein-protein interaction with potential for specific blockade of GPCR desensitization. ^