Variable Response to Antifibrinolytics Correlates with Blood-loss and Transfusion in Posterior Spinal Fusion.

PURPOSE: Posterior spinal fusion (PSF) activates the fibrinolytic protease plasmin, which is implicated in blood loss and transfusion. While antifibrinolytic drugs have improved blood loss and reduced transfusion, variable blood loss has been observed in similar PSF procedures treated with the same dose of antifibrinolytics. However, both the cause of this and the appropriate measures to determine antifibrinolytic efficacy during high-blood-loss spine surgery are unknown, making clinical trials to optimize antifibrinolytic dosing in PSF difficult. We hypothesized that patients undergoing PSF respond differently to antifibrinolytic dosing, resulting in variable blood loss, and that specific diagnostic markers of plasmin activity will accurately measure the efficacy of antifibrinolytics in PSF. METHODS: A prospective study of 17 patients undergoing elective PSF with the same dosing regimen of TXA was conducted. Surgery-induced plasmin activity was exhaustively analyzed in perioperative blood samples and correlated to measures of inflammation, bleeding, and transfusion. RESULTS: While markers of in vivo plasmin activation (PAP and D-dimer) suggested significant breakthrough plasmin activation and fibrinolysis (P < 0.01), in vitro plasmin assays, including TEG, did not detect plasmin activation. In vivo measures of breakthrough plasmin activation correlated with blood loss (R2 = 0.400, 0.264; P < 0.01), transfusions (R2 = 0.388; P < 0.01), and complement activation (R2 = 0.346, P < 0.05). CONCLUSIONS: Despite all patients receiving a high dose of TXA, its efficacy among patients was variable, indicated by notable intra-operative plasmin activity. Markers of in vivo plasmin activation best correlated with clinical outcomes. These findings suggest that the efficacy of antifibrinolytic therapy to inhibit plasmin in PSF surgery should be determined by markers of in vivo plasmin activation in future studies. LEVEL OF EVIDENCE: Level II-diagnostic.

Discovery and Optimization of a Novel Series of Competitive and Central Nervous System-Penetrant Protease-Activated Receptor 4 (PAR4) Inhibitors.

The detailed pharmacology and therapeutic potential of the central PAR4 receptors are poorly understood due to a lack of potent, selective, and brain-penetrant tool compounds. Despite this, robust data with biochemical and genetic tools show the therapeutic potential of PAR4 antagonists in traumatic brain injury, Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders with a neuroinflammatory component. Thus, we performed a functional HTS campaign, identified a fundamentally new PAR4 competitive inhibitor chemotype, optimized this new series (increased potency >45-fold), discovered enantiospecific activity (though opposing preference for human versus mouse PAR4), and engendered high central nervous system penetration (rat Kp's of 0.52 to 4.2 and Kp,uu's of 0.52 to 1.2).

Repurposing of a Thromboxane Receptor Inhibitor Based on a Novel Role in Metastasis Identified by Phenome-Wide Association Study.

Although new drug discoveries are revolutionizing cancer treatments, repurposing existing drugs would accelerate the timeline and lower the cost for bringing treatments to cancer patients. Our goal was to repurpose CPI211, a potent and selective antagonist of the thromboxane A2-prostanoid receptor (TPr), a G-protein-coupled receptor that regulates coagulation, blood pressure, and cardiovascular homeostasis. To identify potential new clinical indications for CPI211, we performed a phenome-wide association study (PheWAS) of the gene encoding TPr, TBXA2R, using robust deidentified health records and matched genomic data from more than 29,000 patients. Specifically, PheWAS was used to identify clinical manifestations correlating with a TBXA2R single-nucleotide polymorphism (rs200445019), which generates a T399A substitution within TPr that enhances TPr signaling. Previous studies have correlated 200445019 with chronic venous hypertension, which was recapitulated by this PheWAS analysis. Unexpectedly, PheWAS uncovered an rs200445019 correlation with cancer metastasis across several cancer types. When tested in several mouse models of metastasis, TPr inhibition using CPI211 potently blocked spontaneous metastasis from primary tumors, without affecting tumor cell proliferation, motility, or tumor growth. Further, metastasis following intravenous tumor cell delivery was blocked in mice treated with CPI211. Interestingly, TPr signaling in vascular endothelial cells induced VE-cadherin internalization, diminished endothelial barrier function, and enhanced transendothelial migration by tumor cells, phenotypes that were decreased by CPI211. These studies provide evidence that TPr signaling promotes cancer metastasis, supporting the study of TPr inhibitors as antimetastatic agents and highlighting the use of PheWAS as an approach to accelerate drug repurposing.

Plasminogen activation in the musculoskeletal acute phase response: Injury, repair, and disease.

The musculoskeletal system is critical for movement and the protection of organs. In addition to abrupt injuries, daily physical demands inflict minor injuries, necessitating a coordinated process of repair referred to as the acute-phase response (APR). Dysfunctional APRs caused by severe injuries or underlying chronic diseases are implicated in pathologic musculoskeletal repair, resulting in decreased mobility and chronic pain. The molecular mechanisms behind these phenomena are not well understood, hindering the development of clinical solutions. Recent studies indicate that, in addition to regulating intravascular clotting, the coagulation and fibrinolytic systems are also entrenched in tissue repair. Although plasmin and fibrin are considered antithetical to one another in the context of hemostasis, in a proper APR, they complement one another within a coordinated spatiotemporal framework. Once a wound is contained by fibrin, activation of plasmin promotes the removal of fibrin and stimulates angiogenesis, tissue remodeling, and tissue regeneration. Insufficient fibrin deposition or excessive plasmin-mediated fibrinolysis in early convalescence prevents injury containment, causing bleeding. Alternatively, excess fibrin deposition and/or inefficient plasmin activity later in convalescence impairs musculoskeletal repair, resulting in tissue fibrosis and osteoporosis, while inappropriate fibrin or plasmin activity in a synovial joint can cause arthritis. Together, these pathologic conditions lead to chronic pain, poor mobility, and diminished quality of life. In this review, we discuss both fibrin-dependent and -independent roles of plasminogen activation in the musculoskeletal APR, how dysregulation of these mechanisms promote musculoskeletal degeneration, and the possibility of therapeutically manipulating plasmin or fibrin to treat musculoskeletal disease.

Curcumin induces secretion of glucagon-like peptide-1 through an oxidation-dependent mechanism.

Curcumin shows antiglycemic effects in animals. Curcumin is chemically unstable at physiological pH, and its oxidative degradation products were shown to contribute to its anti-inflammatory effects. Since the degradation products may also contribute to other effects, we analyzed their role in the antiglycemic activity of curcumin. We quantified curcumin-induced release of glucagon-like peptide 1 (GLP-1) from mouse STC-1 cells that represent enteroendocrine L-cells as a major source of this anti-diabetic hormone. Curcumin induced secretion of GLP-1 in a dose-dependent manner. Two chemically stable analogues of curcumin that do not readily undergo degradation, were less active while two unstable analogues were active secretagogues. Chromatographically isolated spiroepoxide, an unstable oxidative metabolite of curcumin with anti-inflammatory activity, also induced secretion of GLP-1. Stable compounds like the final oxidative metabolite bicyclopentadione, and the major plasma metabolite, curcumin-glucuronide, were inactive. GLP-1 secretion induced by curcumin and its oxidative degradation products was associated with activation of PKC, ERK, and CaM kinase II. Since activity largely correlated with instability of curcumin and the analogues, we tested the extent of covalent binding to proteins in STC-1 cells and found it occurred with similar affinity as N-ethylmaleimide, indicating covalent binding occurred with nucleophilic cysteine residues. These results suggest that oxidative metabolites of curcumin are involved in the antiglycemic effects of curcumin. Our findings support the hypothesis that curcumin functions as a pro-drug requiring oxidative activation to reveal its bioactive metabolites that act by binding to target proteins thereby causing a change in function.

The role of coagulation and platelets in colon cancer-associated thrombosis.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions.

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.

Contributions of Protease-Activated Receptors PAR1 and PAR4 to Thrombin-Induced GPIIbIIIa Activation in Human Platelets.

Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target.

Identification of the minimum PAR4 inhibitor pharmacophore and optimization of a series of 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazoles.

This letter describes the further deconstruction of the known PAR4 inhibitor chemotypes (MWs 490-525 and with high plasma protein binding) to identify a minimum PAR4 pharmacophore devoid of metabolic liabilities and improved properties. This exercise identified a greatly simplified 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazole scaffold that afforded nanomolar inhibition of both activating peptide and γ-thrombin mediated PAR4 stimulation, while reducing both molecular weight and the number of hydrogen bond donors/acceptors by ∼50%. This minimum PAR4 pharmacophore, with competitive inhibition, versus non-competitive of the larger chemotypes, allows an ideal starting point to incorporate desired functional groups to engender optimal DMPK properties towards a preclinical candidate.

Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)-1H-indol-2-yl)methanols as Selective Protease Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and Activity Against γ-Thrombin.

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PAR1, derived from the indole-based 3. Of these, 9j (PAR4 IC50 = 445 nM, PAR1 response IC50 > 30 µM) and 10h (PAR4 IC50 = 179 nM, PAR1 response IC50 > 30 µM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin.

Loss of Serotonin Transporter Function Alters ADP-mediated Glycoprotein αIIbß3 Activation through Dysregulation of the 5-HT2A Receptor.

Reduced platelet aggregation and a mild bleeding phenotype have been observed in patients chronically taking selective serotonin reuptake inhibitors (SSRIs). However, it remains unclear how SSRIs, which inhibit the plasma membrane serotonin transporter (SERT), modulate hemostasis. Here, we examine how sustained inhibition of SERT activity alters serotonergic signaling and influences platelet activation and hemostasis. Pharmaceutical blockade (citalopram dosing) or genetic ablation (SERT(-/-)) of SERT function in vivo led to reduced serotonin (5-hydroxytryptamine (5-HT)) blood levels that paralleled a mild bleeding phenotype in mice. Transfusion of wild-type platelets to SERT(-/-) mice normalized bleeding times to wild-type levels, suggesting that loss of SERTs causes a deficiency in platelet activation. Although SERT(-/-) platelets displayed no difference in P-selectin or αIIbß3 activation upon stimulation with thrombin, ADP-mediated αIIbß3 activation is reduced in SERT(-/-) platelets. Additionally, synergistic potentiation of αIIbß3 activation by ADP and 5-HT is lost in SERT(-/-) platelets. Acute treatment of wild-type platelets with 5-HT2A receptor (5-HT2AR) antagonists or SSRIs revealed that functional 5-HT2ARs, not SERTs, are necessary for the synergistic activation of αIIbß3 by dual 5-HT/ADP stimulation. Pharmacological studies using radiolabeled guanosine 5'-3-O-([(35)S]thio)triphosphate and [(3)H]ketanserin revealed that platelets isolated from SERT(-/-) or citalopram-treated mice have reduced activation of G-proteins coupled to 5-HT2ARs and receptor surface expression. Taken together, these data demonstrate that sustained SERT loss of function reduces 5-HT2AR surface expression that is critical for the synergistic activation of αIIbß3 by 5-HT and ADP. These results highlight an antiplatelet strategy centered on blocking or desensitizing 5-HT2AR to attenuate ADP-mediated αIIbß3 activation.

Platelet Lipidomic Profiling: Novel Insight into Cytosolic Phospholipase A2α Activity and Its Role in Human Platelet Activation.

With a newer, more selective and efficacious cytosolic phospholipase A2α (cPLA2α) inhibitor available, we revisited the role of cPLA2α activity in platelet activation and discovered that a component of platelet signaling, even larger than previously appreciated, relies on this enzyme. In a whole blood shear-based flow chamber assay, giripladib, a cPLA2α inhibitor, reduced platelet adhesion and accumulation on collagen. Moreover, giripladib differentially affected P-selectin expression and GPIIbIIIa activation depending on the agonist employed. While protease-activated receptor 1 (PAR1)-mediated platelet activation was unaffected by giripladib, the levels of PAR4- and GPVI-mediated platelet activation were significantly reduced. Meanwhile, the thromboxane A2 receptor antagonist SQ29548 had no effect on PAR-, GPVI-, or puriniergic receptor-mediated platelet activation, suggesting that another eicosanoid produced downstream of arachidonic acid liberation by cPLA2α was responsible for this large component of PAR4- and GPVI-mediated platelet activation. In parallel, we profiled PAR-mediated changes in glycerophospholipid (GPL) mass with and without giripladib to better understand cPLA2α-mediated lipid metabolism. Phosphatidylcholine and phosphatidylethanolamine (PE) demonstrated the largest consumption of mass during thrombin stimulation. Additionally, we confirm phosphatidylinositol as a major substrate of cPLA2α. A comparison of PAR1- and PAR4-induced metabolism revealed the consumption of more putative arachidonyl-PE species downstream of PAR1 activation. Instead of enhanced cPLA2α activity and therefore more arachidonic acid liberation downstream of PAR4, these results indicate the major role that cPLA2α activity plays in platelet function and suggest that a novel eicosanoid is produced in response to platelet activation that represents a large component of PAR4- and GPVI-mediated responses.

Substituted indoles as selective protease activated receptor 4 (PAR-4) antagonists: Discovery and SAR of ML354.

Herein we report the discovery and SAR of an indole-based protease activated receptor-4 (PAR-4) antagonist scaffold derived from a similarity search of the Vanderbilt HTS collection, leading to MLPCN probe ML354 (VU0099704). Using a novel PAC-1 fluorescent αIIbß3 activation assay this probe molecule antagonist was found to have an IC50 of 140nM for PAR-4 with 71-fold selectivity versus PAR-1 (PAR-1IC50=10µM).

Racial differences in resistance to P2Y12 receptor antagonists in type 2 diabetic subjects.

Although resistance to the P2Y12 antagonist clopidogrel is linked to altered drug metabolism, some studies suggest that these pharmacokinetic abnormalities only partially account for drug resistance. To circumvent pharmacokinetic complications and target P2Y12 receptor function we applied the direct P2Y12 antagonist 2-methylthio-AMP (2-methylthioadenosine 5'-monophosphate triethylammonium salt) to purified platelets ex vivo. Platelets were purified from healthy and type 2 diabetes mellitus (T2DM) patients and stimulated with thrombin or the selective protease-activated receptor agonists, protease-activated receptor 1-activating peptide (PAR1-AP), or PAR4-AP. Platelet activation as measured by αIIbß3 activation, and P-selectin expression was monitored in 141 subjects. Our results demonstrate that, compared with healthy subjects, platelets from diabetic patients are resistant to inhibition by 2-methylthio-AMP, demonstrating P2Y12 pharmacodynamic defects among diabetic patients. Inhibition of thrombin-mediated αIIbß3 activation by 2-methylthio-AMP was lower in diabetic platelets versus healthy platelets. Subgroup analysis revealed a racial difference in the resistance to 2-methylthio-AMP. We found no resistance in platelets from diabetic African Americans; they were inhibited by 2-methylthio-AMP equally as well as platelets from healthy African Americans. In contrast, platelets from Caucasian patients with diabetes were resistant to P2Y12 antagonism compared with healthy Caucasians. Multivariable analysis demonstrated that other variables, such as obesity, age, or gender, could not account for the differential resistance to 2-methylthio-AMP among races. These results suggest that in addition to altered drug metabolism, P2Y12 receptor function itself is altered in the Caucasian diabetic population. The racial difference in platelet function in T2DM is a novel finding, which may lead to differences in treatment as well as new targets for antiplatelet therapy.

Synthesis of indole derived protease-activated receptor 4 antagonists and characterization in human platelets.

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. We sought to develop potent, selective, and novel PAR4 antagonists to test the role of PAR4 in thrombosis and hemostasis. Development of an expedient three-step synthetic route to access a novel series of indole-based PAR4 antagonists also necessitated the development of a platelet based high-throughput screening assay. Screening and subsequent structure activity relationship analysis yielded several selective PAR4 antagonists as well as possible new scaffolds for future antagonist development.

The angiotensin II type 1 receptor C-terminal Lys residues interact with tubulin and modulate receptor export trafficking.

The physiological and pathological functions of angiotensin II are largely mediated through activating the cell surface angiotensin II type 1 receptor (AT1R). However, the molecular mechanisms underlying the transport of newly synthesized AT1R from the endoplasmic reticulum (ER) to the cell surface remain poorly defined. Here we demonstrated that the C-terminus (CT) of AT1R directly and strongly bound to tubulin and the binding domains were mapped to two consecutive Lys residues at positions 310 and 311 in the CT membrane-proximal region of AT1R and the acidic CT of tubulin, suggestive of essentially ionic interactions between AT1R and tubulin. Furthermore, mutation to disrupt tubulin binding dramatically inhibited the cell surface expression of AT1R, arrested AT1R in the ER, and attenuated AT1R-mediated signaling measured as ERK1/2 activation. These data demonstrate for the first time that specific Lys residues in the CT juxtamembrane region regulate the processing of AT1R through interacting with tubulin. These data also suggest an important role of the microtubule network in the cell surface transport of AT1R.

Alpha2B-adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface.

It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α(2B)-adrenergic receptor (α(2B)-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α(2B)-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α(2B)-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α(2B)-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α(2B)-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules.

ADP-ribosylation factors modulate the cell surface transport of G protein-coupled receptors.

ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of alpha(2B)-adrenergic receptor (AR), beta(2)-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that alpha(2B)-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the alpha(2B)-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.

A single conserved leucine residue on the first intracellular loop regulates ER export of G protein-coupled receptors.

The intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The alpha(2B)-adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates alpha(2B)-AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of alpha(2B)-AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of beta(2)-AR, alpha(1B)-AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.