cAMP-dependent protein kinase and heterologous desensitization of the adenylyl cyclase

Previous experiments had shown no differences in desensitization in cells with mutations of the adenylyl cyclase or the cAMP-dependent protein kinase and had ruled out this kinase as a mediator of desensitization; however, the assays of adenylyl cyclase had been made at high concentrations of free magnesium. The work presented in this dissertation documents a role for cAMP-dependent protein kinase which became apparent with assays at low concentrations of free magnesium. (1) The adenylyl cyclase in membranes from wild type S49 lymphoma cells showed substantial desensitization after incubation of the intact cells with low concentrations of epinephrine (5-20 nM). This desensitization was heterologous, that is it reduced the subsequent responses of the adenylyl cyclase to both epinephrine and prostaglandin-E$\sb1$. (2) The adenylyl cyclase in membranes of S49 cyc$\sp-$ cells, which do not make cAMP in response to hormones, and S49 kin$\sp-$ cells, which lack cAMP-dependent protein kinase activity, showed no heterologous desensitization following incubation of the intact cells with low concentrations of hormones. (3) Heterologous desensitization of the adenylyl cyclase was induced by incubations of wild type cells with forskolin, which activates the adenylyl cyclase downstream of the hormone receptors, or dibutyryl-cAMP, which activates the cAMP-dependent protein kinase directly. (4) Site-directed mutagenesis was used to delete the cAMP-dependent protein kinase consensus phosphorylation sequences on the $\beta$-adrenergic receptor. Heterologous desensitization occurred in intact L-cells expressing the wild type receptor or the receptor lacking the C-terminal phosphorylation site; however, only homologous desensitization occurred when the phosphorylation site on the third intracellular loop of the receptor was deleted. (5) To test directly the effects of cAMP-dependent protein kinase on the adenylyl cyclase the catalytic subunit of the kinase was purified from bovine heart and incubated with adenylyl cyclase in plasma membrane preparations. In this cell-free system the kinase caused rapid heterlogous reductions of the responsiveness of the S49 wild type adenylyl cyclase. Additionally, the adenylyl cyclase in kin$\sp-$ membranes, which showed only homologous desensitization in the intact cell, was desensitization by cell-free incubation with the kinase.^ The epinephrine responsiveness was not affected in L-cell membranes expressing the $\beta$-adrenergic receptor lacking the cAMP-dependent protein kinase consensus sequence on the third intracellular loop. ^

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.) ^

$\beta\sb2$-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.) ^

Estrogen induced desensitization of c -{\it fos\/} messenger RNA expression {\it in vivo\/}

In this thesis, we investigated the regulation of the nuclear proto-oncogene, c-fos by estrogen in vivo. In the uterus, estrogen causes a rapid, dramatic and transient induction of c-fos mRNA and this occurs by transcriptional activation. We have discovered a previously unrecognized regulatory mechanism by which fos becomes desensitized to estrogen following the transient induction. We investigated three aspects of this desensitization: (1) the kinetics and general characteristics of the phenomenon; (2) the molecular mechanism of the desensitization; and (3) the relationship of desensitization to estrogen stimulated DNA synthesis. The desensitization occurs between 3-24 hours after initial hormonal stimulation and is reversible within 72 hours. The desensitization is not species specific, in that it occurs in both the rat and mouse. The desensitization also occurs in at least two estrogen responsive tissues, the uterus and vagina. The desensitization is not unique to c-fos, since both c-myc and c-jun show similar patterns of desensitization. However, the desensitization is not observed with creatine kinase B (CKB), indicating that not all estrogen inducible genes become desensitized. In the second general area, we determined the desensitization is at the transcriptional level. The desensitization is homologous, but not heterologous, since estrogen induction does not desensitize c-fos to other agents. Other studies show that the desensitization is not due to the lack of functional estrogen receptors. Taken together, these findings suggest that the desensitization occurs at the level of the estrogen responsive element. In the third major area, we demonstrated that the desensitization appears to be related to estrogen induced DNA synthesis. Support for this suggestion comes from the observation that short acting estrogens which induce fos, but not DNA synthesis, do not produce desensitization. ^

AMPA RECEPTOR ALLOSTERISM: MEASUREMENT OF THE CONFORMATIONAL CHANGES IN THE LIGAND BINDING DOMAIN OF A FUNCTIONAL RECEPTOR

Ionotropic glutamate receptors are important excitatory neurotransmitter receptors in the mammalian central nervous system that have been implicated in a number of neuropathologies such as epilepsy, ischemia, and amyotrophic lateral sclerosis. Glutamate binding to an extracellular ligand binding domain initiates a series of structural changes that leads to the formation of a cation selective transmembrane channel, which consequently closes due to desensitization of the receptor. The crystal structures of the AMPA subtype of the glutamate receptor have been particularly useful in providing initial insight into the conformational changes in the ligand binding domain; however, these structures are limited by crystallographic constraint. To gain a clear picture of how agonist binding is coupled to channel activation and desensitization, it is essential to study changes in the ligand binding domain in a dynamic, physiological state. In this dissertation, a technique called Luminescence Resonance Energy Transfer was used to determine the conformational changes associated with activation and desensitization in a functional AMPA receptor (ÄN*-AMPA) that contains the ligand binding domain and transmembrane segments; ÄN*-AMPA has been modified such that fluorophores can be introduced at specific sites to serve as a readout of cleft closure or to establish intersubunit distances. Previous structural studies of cleft closure of the isolated ligand binding domain in conjunction with functional studies of the full receptor suggest that extent of cleft closure correlates with extent of activation. Here, LRET has been used to show that a similar relationship between cleft closure and activation is observed in the “full length” receptor showing that the isolated ligand binding domain is a good model of the domain in the full length receptor for changes within a subunit. Similar LRET investigations were used to study intersubunit distances specifically to probe conformational changes between subunits within a dimer in the tetrameric receptor. These studies show that the dimer interface is coupled in the open state, and decoupled in the desensitized state, similar to the isolated ligand binding domain crystal structure studies. However, we show that the apo state dimer interface is not pre-formed as in the crystal structure, hence suggesting a mechanism for functional transitions within the receptor based on LRET distances obtained.

THE ROLE OF TYROSINE PHOSPHORYLATION IN THE FUNCTIONS OF THE TUMOR SUPPRESSOR GPRC5A

The retinoic acid inducible G protein coupled receptor family C group 5 type A (GPRC5A) is expressed preferentially in normal lung tissue but its expression is suppressed in the majority of human non-small cell lung cancer cell lines and tissues. This differential expression has led to the idea that GPRC5A is a potential tumor suppressor. This notion was supported by the finding that mice with a deletion of the Gprc5a gene develop spontaneous lung tumors. However, there are various tumor cell lines and tissue samples, including lung, that exhibit higher GPRC5A expression than normal tissues and some reports by other groups that GPRC5A transfection increased cell growth and colony formation. Obviously, GPRC5A has failed to suppress the development of the tumors and the growth of the cell lines where its expression is not suppressed. Since no mutations were detected in the coding sequence of GPRC5A in 20 NSCLC cell lines, it’s possible that GPRC5A acts as a tumor suppressor in the context of some cells but not in others. Alternatively, we raised the hypothesis that the GPRC5A protein may be inactivated by posttranslational modification(s) such as phosphorylation. It is well established that Serine/Threonine phosphorylation of G protein coupled receptors leads to their desensitization and in a few cases Tyrosine phosphorylation of GPCRs has been linked to internalization. Others reported that GPRC5A can undergo tyrosine phosphorylation in the cytoplasmic domain after treatment of normal human mammary epithelial cells (HMECs) with epidermal growth factor (EGF) or Heregulin. This suggested that GPRC5A is a substrate of EGFR. Therefore, we hypothesized that tyrosine phosphorylation of GPRC5A by activation of EGFR signaling may lead to its inactivation. To test this hypothesis, we transfected human embryo kidney (HEK) 293 cells with GPRC5A and EGFR expression vectors and confirmed that GPRC5A can be tyrosine phosphorylated after activation of EGFR by EGF. Further, we found that EGFR and GPRC5A can interact either directly or through other proteins and that inhibition of the EGFR kinase activity decreased the phosphorylation of GPRA5A and the interaction between GPRC5A and EGFR. In c-terminal of GPRC5A, There are four tyrosine residues Y317, Y320, Y347, Y350. We prepared GPRC5A mutants in which all four tyrosine residues had been replaced by phenylalanine (mutant 4F) or each individual Tyr residue was replaced by Phe and found that Y317 is the major site for EGFR mediated phosphorylation in the HEK293T cell line. We also found that EGF can induce GPRC5A internalization both in H1792 transient and stable cell lines. EGF also partially inactivates the suppressive function of GPRC5A on cell invasion activity and anchorage-independent growth ability of H1792 stable cell lines. These finding support our hypothesis that GPRC5A may be inactivated by posttranslational modification- tyrosine phosphorylation.

CONFORMATIONAL CHANGES IN THE EXTRACELLULAR DOMAIN OF GLUTAMATE RECEPTORS

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning. Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic neuron. They receive the burst of neurotransmitters and respond by fielding the neurotransmitters and opening ion channels. Glutamate receptors have been implicated in a number of neuropathologies like ischemia, stroke and amyotrophic lateral sclerosis. Specifically, the NMDA subtype of glutamate receptors has been linked to the onset of Alzheimer’s disease and the subsequent degeneration of neuronal cells. While crystal structures of AMPA and kainate subtypes of glutamate receptors have provided valuable information regarding the assembly and mechanism of activation; little is known about the NMDA receptors. Even the basic question of receptor assembly still remains unanswered. Therefore, to gain a clear understanding of how the receptors are assembled and how agonist binding gets translated to channel opening, I have used a technique called Luminescence Resonance Energy Transfer (LRET). LRET offers the unique advantage of tracking large scale conformational changes associated with receptor activation and desensitization. In this dissertation, LRET, in combination with biochemical and electrophysiological studies, were performed on the NMDA receptors to draw a correlation between structure and function. NMDA receptor subtypes GluN1 and GluN2A were modified such that fluorophores could be introduced at specific sites to determine their pattern of assembly. The results indicated that the GluN1 subunits assembled across each other in a diagonal manner to form a functional receptor. Once the subunit arrangement was established, this was used as a model to further examine the mechanism of activation in this subtype of glutamate receptor. Using LRET, the correlation between cleft closure and activation was tested for both the GluN1 and GluN2A subunit of the NMDA receptor in response to agonists of varying efficacies. These investigations revealed that cleft closure plays a major role in the mechanism of activation in the NMDA receptor, similar to the AMPA and kainate subtypes. Therefore, suggesting that the mechanism of activation is conserved across the different subtypes of glutamate receptors.

Role of protein kinase C in short-term sensitization of {\it Aplysia}

Plasticity at the connections between sensory neurons and their follower cells in Aplysia has been used extensively as a model system to examine mechanisms of simple forms of learning, such as sensitization. Sensitization is induced, at least in part, by the transmitter serotonin (5-HT) and expressed in several forms, including facilitation of sensorimotor connections. Spike broadening has been believed to be a key mechanism underlying facilitation of nondepressed synapses. Previously, this broadening was believed to be dependent primarily on cAMP/protein kinase A (PKA)-mediated reduction of a noninactivating, relatively voltage-independent K$\sp{+}$ current termed the S-K$\sp+$ current (I$\sb{\rm K{,}S}$). Recent evidence, however, suggests that 5-HT-induced somatic spike broadening is composed of at least two components: a cAMP-dependent, rapidly developing component and a cAMP-independent, slowly developing component.^ Phorbol esters, activators of protein kinase C (PKC), mimicked the cAMP-independent component of 5-HT-induced broadening. Staurosporine, which inhibits PKC, had little effect on the rapidly developing component of 5-HT-induced broadening, but inhibited significantly the slowly developing component. These results suggest that PKC is involved in the cAMP-independent component of 5-HT-induced broadening. The membrane currents responsible for the slowly developing component of broadening were examined. Activation of PKC mimicked, and partially occluded, 5-HT-induced modulation of membrane currents above 0 mV, where a voltage-dependent K$\sp+$ current (I$\sb{\rm K{,}V}$) is significantly activated. This modulation was complex because it was associated with a reduction in the magnitude of I$\sb{\rm K{,}V}$, as well as a slowing of both activation and inactivation kinetics of I$\sb{\rm K{,}V}$. These results support the hypothesis that PKC modulates I$\sb{\rm K{,}V}$ and that this modulation contributes to the slowly developing component of 5-HT-induced broadening. Based on these results and others, a new scheme for 5-HT-induced spike broadening is proposed in which the modulatory effects are mediated via two second messenger/protein kinase systems converging and diverging on multiple ionic conductances.^ The relationship between spike broadening and synaptic facilitation was also examined. Pharmacological reduction of I$\sb{\rm K{,}V}$ by low concentrations of 4-aminopyridine (4-AP) led to spike broadening and facilitation of the nondepressed sensorimotor connections, indicating that spike broadening via the reduction of I$\sc{K,V}$ can facilitate the synaptic connection. Further analyses, however, revealed that 4-AP-induced facilitation has qualitative differences from 5-HT- and PKC-induced facilitation. These results suggest that 5-HT- and PKC-induced facilitation of nondepressed synapses is mediated, at least in part, by spike-duration independent (SDI) processes. Under certain conditions, the PKC inhibitor, staurosporine, significantly inhibited the 5-HT-induced facilitation of sensorimotor connections.^ Finally, it was found that activation of PKC increased a basal level of cAMP and that PKC caused desensitization of the 5-HT receptor, which may be a possible negative feedback mechanism through which an extracellular ligand, 5-HT, is regulated. These results suggest that these two second messenger/protein kinase pathways can interact in the sensory neuron. Thus, neuronal plasticity that may contribute to learning and memory appears to involve several complex and interactive processes. ^

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. ^

Regulation of the gastrin-releasing peptide/bombesin receptor

Gastrin-releasing peptide (GRP) and other bombesin-like peptides stimulate hormone secretion and cell proliferation by binding to specific G-protein-coupled receptors. Three studies were performed to identify potential mechanisms involved in GRP/bombesin receptor regulation.^ Although bombesin receptors are localized throughout the gastrointestinal tract, few gastrointestinal cell lines are available to study bombesin action. In the first study, the binding and function of bombesin receptors in the human HuTu-80 duodenal cancer cell line were characterized. ($\sp{125}$I-Tyr$\sp4$) bombesin bound with high affinity to a GRP-preferring receptor. Bombesin treatment increased IP$\sb3$ production, but had no effect on cell proliferation. Similar processing of ($\sp{125}$I-Tyr$\sp4$) bombesin and of GRP-receptors was observed in HuTu-80 cells and Swiss 3T3 fibroblasts, a cell line which mitogenically responds to bombesin. Therefore, the lack of a bombesin mitogenic effect in HuTu-80 cells is not due to unusual processing of ($\sp{125}$I-Tyr$\sp4$) bombesin or rapid GRP-receptor down-regulation.^ In the second study, a bombesin antagonist was developed to study the processing and regulatory events after antagonist binding. As previously shown, receptor bound agonist, ($\sp{125}$I-Tyr$\sp4$) bombesin, was rapidly internalized and degraded in chloroquine-sensitive compartments. Interestingly, receptor-bound antagonist, ($\sp{125}$I-D-Tyr$\sp6$) bombesin(6-13)PA was not internalized, but degraded at the cell-surface. In contrast to bombesin, (D-Tyr$\sp6$) bombesin(6-13)PA treatment did not cause receptor internalization. Together these results demonstrate that receptor regulation and receptor-mediated processing of antagonist is different from that of agonist.^ Bombesin receptors undergo acute desensitization. By analogy to other G-protein-coupled receptors, a potential desensitization mechanism may involve receptor phosphorylation. In the final study, $\sp{32}$P-labelled Swiss 3T3 fibroblasts and CHO-mBR1 cells were treated with bombesin and the GRP-receptor was immunoprecipitated. In both cell lines, bombesin treatment markedly stimulated GRP-receptor phosphorylation. Furthermore, bombesin-stimulated GRP-receptor phosphorylation occurred within the same time period as bombesin-stimulated desensitization, demonstrating that these two processes are correlated.^ In conclusion, these studies of GRP-receptor regulation further our understanding of bombesin action and provide insight into G-protein-coupled receptor regulation in general. ^

The role of beta-arrestin 2 in lysophosphatidic acid-induced NF-kappaB activation

Lysophosphatidic acid (LPA) is a bioactive phospholipid and binds to its receptors, a family of G protein-coupled receptors (GPCR), which initiates multiple signaling cascades and leads to activation of several transcription factors, including NF-κB. NF-κB critically regulates numerous gene expressions, and is persistently active in many diseases. In our previous studies, we have demonstrated that LPA-induced NF-κB activation is dependent on a novel scaffold protein, CARMA3. However, how CARMA3 is recruited to receptor remains unknown. β-Arrestins are a family of proteins involved in desensitization of GPCR signaling. Additionally, β-arrestins function as signaling adaptor proteins, and mediate multiple signaling pathways. Therefore, we have hypothesized that β-arrestins may link CARMA3 to LPA receptors, and facilitate LPA-induced NF-κB activation. ^ Using β-arrestin-deficient MEFs, we found that β-arrestin 2, but not β-arrestin 1, was required for LPA-induced NF-κB activation. Also, we showed that the expression of NF-κB-dependent cytokines, such as interlukin-6, was impaired in β-arrestin 2-deficient MEFs. Mechanistically, we demonstrated the inducible association of endogenous β-arrestin 2 and CARMA3, and we found the CARD domain of CARMA3 interacted with 60-320 residues of β-arrestin 2. To understand why β-arrestin 2, but not β-arrestin 1, mediated NF-κB activation, we generated β-arrestin mutants. However, some mutants degraded quickly, and the rest did not rescue NF-κB activation in β-arrestin-deficient MEFs, though they had similar binding affinities with CARMA3. Therefore, it indicates that slight changes in residues may determine the different functions of β-arrestins. Moreover, we found β-arrestin 2 deficiency impaired LPA-induced IKK kinase activity, while it did not affect LPA-induced IKKα/β phosphorylation. ^ In summary, our results provide the genetic evidence that β-arrestin 2 serves as a positive regulator in NF-κB signaling pathway by connecting CARMA3 to LPA receptors. Additionally, we demonstrate that β-arrestin 2 is required for IKKα/β activation, but not for the inducible phosphorylation of IKKα/β. Because the signaling pathways around the membrane-proximal region of LPA receptors and GPCRs are quite conserved, our results also suggest a possible link between other GPCRs and CARMA3-mediated NF-κB activation. To fully define the role of β-arrestins in LPA-induced NF-κB signaling pathways will help to identify new drug targets for clinical therapeutics.^

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. ^

TRPV1 Channels Contribute to Behavioral Hypersensitivity and Spontaneous Activity in Nociceptors After Spinal Cord Injury

A majority of persons who have sustained spinal cord injury (SCI) develop chronic pain. While most investigators have assumed that the critical mechanisms underlying neuropathic pain after SCI are restricted to the central nervous system (CNS), recent studies showed that contusive SCI results in a large increase in spontaneous activity in primary nociceptors, which is correlated significantly with mechanical allodynia and thermal hyperalgesia. Upregulation of ion channel transient receptor vanilloid 1 (TRPV1) has been observed in the dorsal horn of the spinal cord after SCI, and reduction of SCI-induced hyperalgesia by a TRPV1 antagonist has been claimed. However, the possibility that SCI enhances TRPV1 expression and function in nociceptors has not been tested. I produced contusive SCI at thoracic level T10 in adult, male rats and harvested lumbar (L4/L5) dorsal root ganglia (DRG) from sham-treated and SCI rats 3 days and 1 month after injury, as well as from age-matched naive control rats. Whole-cell patch clamp recordings were made from small (soma diameter <30 >μm) DRG neurons 18 hours after dissociation. Capsaicin-induced currents were significantly increased 1 month, but not 3 days, after SCI compared to neurons from control animals. In addition, Ca2+ transients imaged during capsaicin application were significantly greater 1 month after SCI. Western blot experiments indicated that expression of TRPV1 protein in DRG is also increased 1 month after SCI. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hindlimb withdrawal responses to heat and mechanical stimuli. Similar reversal of behavioral hypersensitivity was induced by intrathecal delivery of oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of spontaneous activity in dissociated nociceptors after SCI. Limited activation of TRPV1 was found to induce prolonged repetitive firing without accommodation or desensitization, and this effect was enhanced by SCI. These data suggest that SCI enhances TRPV1 expression and function in primary nociceptors, increasing the excitability and spontaneous activity of these neurons, thus contributing to chronic pain after SCI.