Adrenomedullin improves fertility and promotes pinopodes and cell junctions in the peri-implantation endometrium.

Implantation is a complex event demanding contributions from both embryo and endometrium. Despite advances in assisted reproduction, endometrial receptivity defects persist as a barrier to successful implantation in women with infertility. We previously demonstrated that maternal haploinsufficiency for the endocrine peptide adrenomedullin (AM) in mice confers a subfertility phenotype characterized by defective uterine receptivity and sparse epithelial pinopode coverage. The strong link between AM and implantation suggested the compelling hypothesis that administration of AM prior to implantation may improve fertility, protect against pregnancy complications, and ultimately lead to better maternal and fetal outcomes. Here, we demonstrate that intrauterine delivery of AM prior to blastocyst transfer improves the embryo implantation rate and spacing within the uterus. We then use genetic decrease-of-function and pharmacologic gain-of-function mouse models to identify potential mechanisms by which AM confers enhanced implantation success. In epithelium, we find that AM accelerates the kinetics of pinopode formation and water transport and that, in stroma, AM promotes connexin 43 expression, gap junction communication, and barrier integrity of the primary decidual zone. Ultimately, our findings advance our understanding of the contributions of AM to uterine receptivity and suggest potential broad use for AM as therapy to encourage healthy embryo implantation, for example, in combination with in vitro fertilization.

Reproductive physiology of a humanized GnRH receptor mouse model: application in evaluation of human-specific analogs.

The human GnRH receptor (GNRHR1) has a specific set of properties with physiological and pharmacological influences not appropriately modeled in laboratory animals or cell-based systems. To address this deficiency, we have generated human GNRHR1 knock-in mice and described their reproductive phenotype. Measurement of pituitary GNRHR1 transcripts from homozygous human GNRHR1 knock-in (ki/ki) mice revealed a severe reduction (7- to 8-fold) compared with the mouse Gnrhr1 in wild-type mice. ¹²5I-GnRH binding assays on pituitary membrane fractions corroborated reduced human GNRHR1 protein expression in ki/ki mice, as occurs with transfection of human GNRHR1 in cell lines. Female homozygous knock-in mice displayed normal pubertal onset, indicating that a large reduction in GNRHR1 expression is sufficient for this process. However, ki/ki females exhibited periods of prolonged estrous and/or metestrous and reduced fertility. No impairment was found in reproductive maturity or adult fertility in male ki/ki mice. Interestingly, the serum LH response to GnRH challenge was reduced in both knock-in males and females, indicating a reduced GNRHR1 signaling capacity. Small molecules targeting human GPCRs usually have poor activities at homologous rodent receptors, thus limiting their use in preclinical development. Therefore, we tested a human-specific GnRH1 antagonist, NBI-42902, in our mouse model and demonstrated abrogation of a GnRH1-induced serum LH rise in ki/ki mice and an absence of effect in littermates expressing the wild-type murine receptor. This novel model provides the opportunity to study the human receptor in vivo and for screening the activity of human-specific GnRH analogs.

Pharmacological, Pharmacokinetic, Pharmacodynamic and Physicochemical Characterization of FE 205030: A Potent, Fast Acting, Injectable CGRP Receptor Antagonist for the Treatment of Acute Episodic Migraine.

The neuropeptide calcitonin gene-related peptide (CGRP) is known to play a central role in the underlying pathophysiology of migraine. In comparison to the effective triptan class of antimigraine treatments, the CGRP antagonists possess a comparable efficacy but a superior cardiovascular safety profile in patients. This paper describes the development of selective and potent peptidic CGRP antagonist, FE 205030, that has a fast onset of action and an optimal half-life (subcutaneous Tmax ~ 60 min, and t1/2 ~ 4.4 h in 80 kg pigs, respectively), which is key to prevention of the progression of debilitating migraine symptoms. The in vivo efficacy of this agent has been established a translational pharmacodynamic model (inhibition of capsaicin-induced increase in skin blood flow) in cynomolgus monkeys and shows maximal inhibitory activity at circulating concentrations of 30-100 nM. Antagonist activity of FE 205030 was characterized on CGRP-induced vasodilation in isolated human mesenteric resistance arteries in an ex vivo isometric myograph study, and FE 205030 effectively blocked CGRP-induced vasodilation with a pA2 of 9.3 ± 0.1, mean ± standard error. Multispecies allometric scaling and modeling of subcutaneous (SC) effective concentrations indicates that a dose of 10-30 mg/day is sufficient to achieve a drug exposure/target coverage of 8h, which is useful to prevent migraine recurrence in patients. The molecule also possesses appropriate physicochemical properties that allows for a convenient dosing form factor of 1 ml injection volume with a sufficient solubility and acceptable short-term stability, optimal for treatment of acute migraine episodes in patients. Hence, FE 205030 may provide an important fast-acting injectable option for patients suffering from frequent acute migraine episodes, complementary to preventative monoclonal antibodies and oral small molecule CGRP-R antagonist therapies.

Cabergoline Stimulates Human Endometrial Stromal Cell Decidualization and Reverses Effects of Interleukin-1ß In Vitro.

CONTEXT: Human embryonic implantation is regulated by neuroendocrine hormones, ovarian steroids, growth factors, and cytokines. Sympathetic innervation of the uterus also may play a role. OBJECTIVE: We tested the hypothesis that cabergoline (Cb), an agonist of type 2 dopamine receptors (DRD2), could influence endometrial decidualization in vitro. METHODS: Immunohistochemistry confirmed the presence of catecholaminergic neurons in human uterine tissue. DRD2 mRNA and protein expression in endometrial tissue and cells were validated by quantitative RT-PCR, cDNA microarrays, RNA sequencing, and Western blotting. Isolated human endometrial stromal cells (ESC) were subjected to dose-response and time-course experiments in the absence or presence of decidualizing hormones (10 nM estradiol, 100 nM progesterone, and 0.5 mM dibutyryl cAMP). In some cases, interleukin (IL)-1ß (0.1 nM) was used as an inflammatory stimulus. Well-characterized in vitro biomarkers were quantified. RESULTS: DRD2 were maximally expressed in vivo in the mid-secretory phase of the cycle and upregulated in ESC in response to decidualizing hormones, as were classical (eg, prolactin) and emerging (eg, VEGF and connexin 43) differentiation biomarkers. Cabergoline treatment more than doubled decidual biomarker expression, whereas risperidone, a dopamine receptor antagonist, inhibited ESC differentiation by >50%. Cabergoline induced characteristic decidual morphology changes and blocked detrimental effects of IL-1ß on decidual cytology. CONCLUSION: Our results support the hypothesis that dopaminergic neurons modulate decidualization in situ. We postulate that dopamine agonists, like Cb, could be developed as therapeutic agents to enhance implantation in couples with inflammation-associated infertility.

Additive effect of simultaneous continuous administration of degarelix and TAK-448 on LH suppression in a castrated rat model.

Gonadotropin releasing hormone (GnRH) analogs have long been used in androgen deprivation therapy (ADT) in the treatment of prostate cancer. Chronic administration of either GnRH agonists or antagonists leads to suppression of testosterone production in the testes via either downregulation or direct blockade of the GnRH receptor in the pituitary, respectively. Chronic administration of kisspeptin analogs has more recently been shown to lead to testosterone suppression via desensitization of GnRH neurons in the hypothalamus and an optimized kisspeptin analog, TAK-448, was proven effective in a small phase 1 trial. The current study explored the hypothesis that co-administration of TAK-448 and the GnRH antagonist, degarelix, would have an additive effect on hormonal suppression, as a result of simultaneous intervention in separate steps in the same pathway. TAK-448 or degarelix were first administered individually to castrated rats in order to identify low doses capable of partial or no suppression of luteinizing hormone (LH). In the second step, combinations of the low doses of TAK-448 and degarelix were assessed in a 14 day study and compared to the drugs administered separately. The results showed that simultaneous intervention at the kisspeptin and GnRH receptors caused a more pronounced LH suppression than either drug alone, demonstrating an additive or potentiating effect. These results suggest that such a drug combination may hold promise as novel forms of androgen deprivation therapy in the treatment of prostate cancer.

Pharmacological characterization of a novel nonpeptide antagonist of the human gonadotropin-releasing hormone receptor, NBI-42902.

Suppression of the hypothalamic-pituitary-gonadal axis by peptides that act at the GnRH receptor has found widespread use in clinical practice for the management of sex-steroid-dependent diseases (such as prostate cancer and endometriosis) and reproductive disorders. Efforts to develop orally available GnRH receptor antagonists have led to the discovery of a novel, potent nonpeptide antagonist, NBI-42902, that suppresses serum LH concentrations in postmenopausal women after oral administration. Here we report the in vitro and in vivo pharmacological characterization of this compound. NBI-42902 is a potent inhibitor of peptide radioligand binding to the human GnRH receptor (K(i) = 0.56 nm). Tritiated NBI-42902 binds with high affinity (K(d) = 0.19 nm) to a single class of binding sites and can be displaced by a range of peptide and nonpeptide GnRH receptor ligands. In vitro experiments demonstrate that NBI-42902 is a potent functional, competitive antagonist of GnRH stimulated IP accumulation, Ca(2+) flux, and ERK1/2 activation. It did not stimulate histamine release from rat peritoneal mast cells. Finally, it is effective in lowering serum LH in castrated male macaques after oral administration. Overall, these data provide a benchmark of pharmacological characteristics required for a nonpeptide GnRH antagonist to effectively suppress gonadotropins in humans and suggest that NBI-42902 may have clinical utility as an oral agent for suppression of the hypothalamic-pituitary-gonadal axis.

Trypanosoma cruzi antigen that interacts with the beta1-adrenergic receptor and modifies myocardial contractile activity.

Previously, we have demonstrated that plasma membranes from the parasite Trypanosoma cruzi (T. cruzi) recognize and adhere to host cells through parasite surface attachment molecules that have affinity for beta(1)-adrenergic receptors (beta(1)-ARs) on target organs. In this report we identify a parasite protein that not only interacts with beta(1)-ARs, but also displays beta-agonist-like activity. We demonstrate that a recombinant maltose binding protein fusion of Tc13 Tul (MBP-Tc13 Tul), a member of the T. cruzi antigen 13 family of surface antigen proteins, competes for binding sites with the beta-adrenergic receptor antagonist [125I]-CYP on membranes purified both from CHO cells expressing human beta(1)-ARs and from rat atria. The competition is prevented by pre-treating MBP-Tc13 Tul with antibodies directed against the EPKSA repeat domain of Tc13 Tul, implicating this portion of the molecule in binding to the beta(1)-AR. Furthermore, MBP-Tc13 Tul activates rat myocardial beta(1)-ARs, resulting in synthesis of cyclic adenosine monophosphate (cAMP) and an increase in cardiac contractility. These biological effects are selectively suppressed by the beta(1)-AR antagonist atenolol, by a synthetic peptide corresponding to the second extracellular loop of the human beta(1)-AR, and by the anti-EPKSA repeat antibodies. These results imply that the Tc13 Tul cell-surface antigen of T. cruzi plays a central role in misregulating the beta(1)-AR following parasite infection, and may be a causative factor of dysautonomic syndrome described in Chagas' disease.

Validation and application of a liquid chromatography-tandem mass spectrometric method for the simultaneous determination of testosterone and dihydrotestosterone in rat prostatic tissue using a 96-well format.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to extract and quantify the androgen concentration in the rat prostate. This method introduced a novel 96-well plate format for the extraction and derivatization of testosterone (T) and dihydrotestosterone (DHT) from rat prostatic tissue that greatly simplified the sample preparation procedure. Due to the difficulty to obtain reproducible specimens with non-detectable level of androgen, a matrix-free standard solution was used for method validation. Both T and DHT calibration curves were linear over the calibration range (12.5-2500 pg) with correlation coefficient values greater than 0.9900. The intra-day and inter-day accuracy, reported as %bias, and precision, reported as %CV, of T and DHT were within +/-10%. The lower limit of detection (LLOD) and lower limits of quantification (LLOQ) for both T and DHT were determined to be 5 and 12.5 pg. The validation results demonstrated the selectivity, sensitivity, accuracy, precision, linearity and ruggedness of the method, as well as the suitability of the method for simultaneous detection of T and DHT in rat prostatic tissues. The validated method was successfully applied to determine the physiological T and DHT level in rat prostatic tissues. Similarly to the serum concentration profile pattern, T and DHT intraprostatic levels peaked 2 h after lights-on and decreased after lights-off with DHT level approximately 4-fold greater than T.

Trapping of a nonpeptide ligand by the extracellular domains of the gonadotropin-releasing hormone receptor results in insurmountable antagonism.

Drugs that exhibit insurmountable antagonism are proposed to provide improved clinical efficacy through extended receptor blockade. Long-term suppression of the gonadotropin-releasing hormone receptor (GnRHR) is an important therapeutic approach for a number of sex hormone-dependent diseases. In this study, we describe the mechanism and structural components required for insurmountable activity of a GnRHR antagonist. TAK-013 behaves as an insurmountable antagonist at the human receptor (hGnRHR) but as a surmountable antagonist at the macaque receptor (mGnRHR). Mutation of the eight residues that differ between hGnRHR and mGnRHR identified Ser-203 and Leu-300 in extracellular loops (ECL) 2 and 3 of hGnRHR as essential for the insurmountability of TAK-013. Substitution of the corresponding residues in mGnRHR with Ser and Leu (mGnRHR-P203S/V300L) converts TAK-013 to an insurmountable antagonist. In addition, mutation of Met-24 to Leu in the amino terminus of hGnRHR also ablates the insurmountable antagonism of TAK-013. The mechanism of insurmountability of TAK-013 was determined to be governed by its rate of dissociation from the receptor. Although the association rates of TAK-013 to hGnRHR, mGnRHR, and mGnRHR-P203S/V300L do not differ, the dissociation rate half-life correlates closely with the degree of insurmountability observed (169, 9, and 55 min, respectively). Taken together, these data suggest a model of the GnRHR in which ECL2, ECL3, and the amino terminus engage with TAK-013 upon its binding to the transmembrane region of the receptor. These additional interactions form a "trap door" above TAK-013, restricting its dissociation and thus resulting in its insurmountability.

beta-Arrestin 2 expression determines the transcriptional response to lysophosphatidic acid stimulation in murine embryo fibroblasts.

G protein-coupled receptors often employ novel signaling mechanisms, such as transactivation of epidermal growth factor (EGF) receptors or G protein-independent signals transmitted by beta-arrestins, to control the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In this study we investigated the role of beta-arrestins in lysophosphatidic acid (LPA) receptor-stimulated ERK1/2 activation using fibroblast lines derived from wild type, beta-arrestin 1, beta-arrestin 2, and beta-arrestin 1/2 knock-out mice. LPA stimulation produced robust ERK1/2 phosphorylation in all four backgrounds. In cells lacking beta-arrestin 2, >80% of LPA-stimulated ERK1/2 phosphorylation was mediated by transactivated EGF receptors. In contrast, ERK1/2 activation in cells expressing beta-arrestin 2 was predominantly EGF receptor-independent. Introducing FLAG epitope-tagged beta-arrestin 2 into the beta-arrestin 1/2 null background restored EGF receptor-independent ERK1/2 activation, indicating that beta-arrestin 2 expression confers ERK1/2 activation via a distinct mechanism. To determine the contributions of beta-arrestin 2, transactivated EGF receptors, and ERK1/2 to LPA-stimulated transcriptional responses, we employed gene expression arrays containing cDNA markers for G protein-coupled receptor-mediated signaling. In the beta-arrestin 1/2 null background, 1 h of exposure to LPA significantly increased transcription of seven marker genes. Six of these responses were EGF receptor-dependent, and two required ERK1/2 activation. In beta-arrestin 2 expressing cells, three of the seven LPA-stimulated transcriptional responses observed in the beta-arrestin 1/2 null background were lost. The four residual responses were independent of EGF receptor transactivation, but all were ERK1/2-dependent. These data indicate that beta-arrestin 2 functions both to attenuate EGF receptor transactivation-dependent signaling and to promote a distinct subset of ERK1/2-mediated responses to LPA receptor activation.

Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling.

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.

Distinct conformations of the corticotropin releasing factor type 1 receptor adopted following agonist and antagonist binding are differentially regulated.

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.

Beta-arrestin1 mediates insulin-like growth factor 1 (IGF-1) activation of phosphatidylinositol 3-kinase (PI3K) and anti-apoptosis.

beta-arrestins (1 and 2) are widely expressed cytosolic proteins that play central roles in G protein-coupled receptor signaling. beta-arrestin1 is also recruited to the insulin-like growth factor 1 (IGF-1) receptor, a receptor tyrosine kinase, upon agonist binding. Here we report that, in response to IGF-1 stimulation, beta-arrestin1 mediates activation of phosphatidylinositol 3-kinase in a pathway that leads to the subsequent activation of Akt and anti-apoptosis. This process is independent of both Gi and ERK activity. The pathway fails in mouse embryo fibroblasts lacking both beta-arrestins and is restored by stable transfection of beta-arrestin1. Remarkably, this pathway is insensitive to chemical inhibition of IGF-1 receptor tyrosine kinase activity. These results suggest that, in addition to their roles in G protein-coupled receptor signaling, beta-arrestins couple the IGF-1 receptor tyrosine kinase to the phosphatidylinositol 3-kinase system and suggest that this mechanism is operative independently of the tyrosine kinase activity of the receptor.

Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway.

Arrestins bind phosphorylated G-protein coupled-receptors (GPCR) and inhibit agonist-induced signal transduction by uncoupling the receptors from their cognate G-proteins. beta-arrestins also act as adaptors that target GPCR to endocytic clathrin-coated vesicles. Unlike cellular GPCRs, the human cytomegalovirus GPCRs and chemokine receptor, US28, shows constitutive signal transduction activity and undergoes constitutive endocytosis. To determine the role of beta-arrestins in US28 trafficking, we used embryonic fibroblasts derived from beta-arrestin knockout mice. In these cells, the internalization of transfected beta2-adrenergic receptor and of the cellular chemokine receptor CCR5 was impaired. By contrast, US28 distribution was unaffected, and US28-mediated RANTES internalization was similar in normal and knockout cell lines. To investigate whether a clathrin-mediated pathway is involved in US28 endocytosis, we developed small interfering RNA against the micro2-adaptin subunit of the AP-2 adaptor complex. In cells transfected with micro2 small interfering RNA transferrin endocytosis was severely inhibited. Antibody-feeding experiments and biochemical analysis showed that US28 internalization was also inhibited. Together, these data indicate that US28 endocytosis occurs via a clathrin-mediated mechanism but is independent of beta-arrestins.

beta -Arrestins regulate protease-activated receptor-1 desensitization but not internalization or Down-regulation.

The widely expressed beta-arrestin isoforms 1 and 2 bind phosphorylated G protein-coupled receptors (GPCRs) and mediate desensitization and internalization. Phosphorylation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, is important for desensitization and internalization, however, the role of beta-arrestins in signaling and trafficking of PAR1 remains unknown. To assess beta-arrestin function we examined signaling and trafficking of PAR1 in mouse embryonic fibroblasts (MEFs) derived from beta-arrestin (betaarr) knockouts. Desensitization of PAR1 signaling was markedly impaired in MEFs lacking both betaarr1 and betaarr2 isoforms compared with wild-type cells. Strikingly, in cells lacking only betaarr1 PAR1 desensitization was also significantly impaired compared with betaarr2-lacking or wild-type cells. In wild-type MEFs, activated PAR1 was internalized through a dynamin- and clathrin-dependent pathway and degraded. Surprisingly, in cells lacking both betaarr1 and betaarr2 activated PAR1 was similarly internalized through a dynamin- and clathrin-dependent pathway and degraded, whereas the beta(2)-adrenergic receptor (beta(2)-AR) failed to internalize. A PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation failed to internalize in both wild-type and beta-arrestin knockout cells. Thus, PAR1 appears to utilize a distinct phosphorylation-dependent but beta-arrestin-independent pathway for internalization through clathrin-coated pits. Together, these findings strongly suggest that the individual beta-arrestin isoforms can differentially regulate GPCR desensitization and further reveal a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of arrestins.

Differential desensitization, receptor phosphorylation, beta-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7.

Many members of the chemokine receptor family of G protein-coupled receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of receptor phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated phosphorylation of the receptor, whereas residues serine 356, 357, 364, and 365 are important for basal receptor phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the phosphorylation sites in the tail of the receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the receptor and activation of ERK1/2, and another that is impaired in these functions.

Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.

It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that beta2AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1/2 thus behaving as dual efficacy ligands. ERK1/2 activation by dual efficacy ligands was not affected by ADP-ribosylation of Galphai and could be observed in S49-cyc- cells lacking Galphas indicating that, unlike the conventional agonist isoproterenol, these drugs induce ERK1/2 activation in a Gs/i-independent manner. In contrast, this activation was inhibited by a dominant negative mutant of beta-arrestin and was abolished in mouse embryonic fibroblasts lacking beta-arrestin 1 and 2. The role of beta-arrestin was further confirmed by showing that transfection of beta-arrestin 2 in these knockout cells restored ICI118551 promoted ERK1/2 activation. ICI118551 and propranolol also promoted beta-arrestin recruitment to the receptor. Taken together, these observations suggest that beta-arrestin recruitment is not an exclusive property of agonists, and that ligands classically classified as inverse agonists rely exclusively on beta-arrestin for their positive signaling activity. This phenomenon is not unique to beta2-adrenergic ligands because SR121463B, an inverse agonist on the V2 vasopressin receptor-stimulated adenylyl cyclase, recruited beta-arrestin and stimulated ERK1/2. These results point to a multistate model of receptor activation in which ligand-specific conformations are capable of differentially activating distinct signaling partners.

Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes. Differential regulation of beta-arrestins 1 and 2.

G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. Using the GPCR subtypes for thyrotropin-releasing hormone (TRHR1 and TRHR2), the aim of this study was to determine if subtype-specific differences exist in the trafficking process. If so, we wished to determine the impact of homo- and hetero-oligomerization on TRHR subtype trafficking as a potential mechanism for the differential cellular responses induced by TRH. Expression of either beta-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only beta-arrestin 2 could enhance TRHR2 internalization. The preference for beta-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a beta-arrestin 2 knockout or a beta-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both beta-arrestins. The differential beta-arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET). Both BRET and confocal microscopy were also used to demonstrate that TRHR subtypes form hetero-oligomers. In addition, these hetero-oligomers have altered internalization kinetics compared with the homo-oligomer. The formation of TRHR1/2 heteromeric complexes increased the interaction between TRHR2 and beta-arrestin 1. This may be due to conformational differences between TRHR1/2 hetero-oligomers versus TRHR2 homo-oligomers as a mutant TRHR1 (TRHR1 C335Stop) that does not interact with beta-arrestins, could also enhance TRHR2/beta-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the beta-arrestins and also provides the first evidence that the interactions of TRHRs with beta-arrestin may be altered by hetero-oligomer formation.

The adaptor protein beta-arrestin2 enhances endocytosis of the low density lipoprotein receptor.

Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor beta-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of beta-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether beta-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, beta-arrestin1 and beta-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by approximately 70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only beta-arrestin2, and not beta-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected beta-arrestin1/beta-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of beta-arrestin2, but not beta-arrestin1, LDLR endocytosis was reduced. Moreover, beta-arrestin2-/- MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, beta-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to beta-arrestin2, and the efficiency of LDLR endocytosis in cells expressing beta-arrestin2 physiologically. We conclude that beta-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.