Characterizing the role of Pdgfra in calvarial development.

BACKGROUND: Mammalian calvarium is composed of flat bones developed from two origins, neural crest, and mesoderm. Cells from both origins exhibit similar behavior but express distinct transcriptomes. It is intriguing to ask whether genes shared by both origins play similar or distinct roles in development. In the present study, we have examined the role of Pdgfra, which is expressed in both neural crest and mesoderm, in specific lineages during calvarial development. RESULTS: We found that in calvarial progenitor cells, Pdgfra is needed to maintain normal proliferation and migration of neural crest cells but only proliferation of mesoderm cells. Later in calvarial osteoblasts, we found that Pdgfra is necessary for both proliferation and differentiation of neural crest-derived cells, but not for differentiation of mesoderm-derived cells. We also examined the potential interaction between Pdgfra and other signaling pathway involved in calvarial osteoblasts but did not identify significant alteration of Wnt or Hh signaling activity in Pdgfra genetic models. CONCLUSIONS: Pdgfra is required for normal calvarial development in both neural crest cells and mesoderm cells, but these lineages exhibit distinct responses to alteration of Pdgfra activity.

Characterizing expression pattern of Six2Cre during mouse craniofacial development.

Craniofacial development is a complex process involving diverse cell populations. Various transgenic Cre lines have been developed to facilitate studying gene function in specific tissues. In this study, we have characterized the expression pattern of Six2Cre mice at multiple stages during craniofacial development. Our data revealed that Six2Cre lineage cells are predominantly present in frontal bone, mandible, and secondary palate. Using immunostaining method, we found that Six2Cre triggered reporter is co-expressed with Runx2. In summary, our data showed Six2Cre can be used to study gene function during palate development and osteogenesis in mouse models.

Pdgfra regulates multipotent cell differentiation towards chondrocytes via inhibiting Wnt9a/beta-catenin pathway during chondrocranial cartilage development.

The mammalian skull is composed of the calvarial bones and cartilages. Malformation of craniofacial cartilage has been identified in multiple human syndromes. However, the mechanisms of their development remain largely unknown. In the present study, we identified Pdgfra as a novel player of chondrocranial cartilage development. Our data show that Pdgfra is required for normal chondrocranial cartilage development. Using tissue-specific genetic tools, we demonstrated that Pdgfra is essential for chondrocyte progenitors formation, but not in mature chondrocytes. Further analysis revealed that Pdgfra regulates chondrocytes progenitors development at two stages: in embryonic mesenchymal stem cells (eMSCs), Pdgfra directs their differentiation toward chondrocyte progenitors; in chondrocytes progenitors, Pdgfra activation promotes cell proliferation. We also found that excessive Pdgfra activity causes ectopic cartilage formation. Our data show that Pdgfra directs eMSCs differentiation via inhibiting Wnt9a transcription and its downstream signaling, and activating Wnt signaling rescues ectopic cartilage phenotype caused by excessive Pdgfra activity. In summary, our study dissected the role of Pdgfra signaling in chondrocranial cartilage formation, and illustrated the underlying mechanisms at multiple stages.

Identification and characterization of the antiplasmodial activity of Hsp90 inhibitors.

BACKGROUND: The recent reduction in mortality due to malaria is being threatened by the appearance of Plasmodium falciparum parasites that are resistant to artemisinin in Southeast Asia. To limit the impact of resistant parasites and their spread across the world, there is a need to validate anti-malarial drug targets and identify new leads that will serve as foundations for future drug development programmes targeting malaria. Towards that end, the antiplasmodial potential of several Hsp90 inhibitors was characterized. Because, the Hsp90 chaperone has been suggested as a good drug target against multiple parasitic infections including malaria. RESULTS: Chemically diverse sets of Hsp90 inhibitors, evaluated in clinical trials as anti-cancer agents, were tested against the malaria parasite. Most of the compounds showed strong antiplasmodial activity in growth inhibition assays against chloroquine sensitive and resistant strains. There was a good agreement between the compound in vitro anti-parasitic activity and their affinity against the Plasmodium chaperone. The two most potent Hsp90 inhibitors also showed cytocidal activity against two P. falciparum strains. Their antiplasmodial activity affected all parasite forms during the malaria blood cycle. However, the compounds activity against the parasite showed no synergy when combined with anti-malarial drugs, like chloroquine or DHA. DISCUSSION: The Hsp90 inhibitors anti-parasitic activity correlates with their affinity to their predicted target the P. falciparum chaperone Hsp90. However, the most effective compounds also showed high affinity for a close homologue, Grp94. This association points to a mode of action for Hsp90 inhibitors that correlate compound efficacy with multi-target engagement. Besides their ability to limit parasite replication, two compounds also significantly impacted P. falciparum viability in vitro. Finally, a structural analysis suggests that the best hit represents a promising scaffold to develop parasite specific leads according. CONCLUSION: The results shown that Hsp90 inhibitors are lethal against the malaria parasite. The correlation between biochemical and in vitro data strongly supports Hsp90 as a drug target against the malaria parasite. Furthermore, at least one Hsp90 inhibitor developed as anticancer therapeutics could serve as starting point to generate P. falciparum-specific lead compounds.

Regulation of transforming growth factor-beta1 (TGF-ß1)-induced pro-fibrotic activities by circadian clock gene BMAL1.

BACKGROUND: BMAL1 is a transcriptional activator of the molecular clock feedback network. Besides its role in generating circadian rhythms, it has also been shown to be involved in the modulation of cell proliferation, autophagy and cancer cell invasion. However, the role of BMAL1 in pulmonary fibrogenesis is still largely unknown. In this study, we investigated the crosstalk between BMAL1 and the signaling transduction and cellular activities of TGF-ß1, a key player in lung fibrogenesis. METHODS: Lungs from wild type and TGF-ß1-adenovirus-infected mice were harvested and homogenized for isolation of RNA and protein. RT-PCR and Western Blotting were employed to measure the expression level of clock genes and TGF-ß1-induced downstream target genes. siRNA against human BMAL1 gene was transfected by using lipofectamine RNAiMAX to knockdown the endogenous BMAL1 in both lung epithelial cells and fibroblasts. RESULTS: Our results showed that TGF-ß1 is able to up-regulate BMAL1 expression in both lung epithelial cells and normal lung fibroblasts. In animal models of pulmonary fibrosis, BMAL1 expression was also significantly higher in adenovirus-TGF-ß1-infected mice than in the control group. Interestingly, BMAL1 was mostly found in a deacetylated form in the presence of TGF-ß1. Importantly, siRNA-mediated knockdown of BMAL1 significantly attenuated the canonical TGF-ß1 signaling pathway and altered TGF-ß1-induced epithelial-mesenchymal transition and MMP9 production in lung epithelial cells. In addition, BMAL1 knockdown inhibited the fibroblast to myofibroblast differentiation of normal human lung fibroblasts. CONCLUSIONS: Our results indicate that activation of TGF-ß1 promotes the transcriptional induction of BMAL1. Furthermore, BMAL1 is required for the TGF-ß1-induced signaling transduction and pro-fibrotic activities in the lung.

A triple arg motif mediates α(2B)-adrenergic receptor interaction with Sec24C/D and export.

Recent studies have demonstrated that cargo exit from the endoplasmic reticulum (ER) may be directed by ER export motifs recognized by components of the coat protein II (COPII) vesicles. However, little is known about ER export motifs and vesicle targeting of the G protein-coupled receptor (GPCR) superfamily. Here, we have demonstrated that a triple Arg (3R) motif in the third intracellular loop functions as a novel ER export signal for α(2B)-adrenergic receptor (α(2B)-AR). The 3R motif mediates α(2B)-AR interaction with Sec24C/D and modulates ER exit, cell surface transport and function of α(2B)-AR. Furthermore, export function of the 3R motif is independent of its position within α(2B)-AR and can be conferred to CD8 glycoprotein. These data provide the first evidence implicating that export of GPCRs is controlled by code-directed interactions with selective components of the COPII transport machinery.

Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers.

G protein-coupled receptors (GPCRs) transmit signals by forming active-state complexes with heterotrimeric G proteins. It has been suggested that some GPCRs also assemble with G proteins before ligand-induced activation and that inactive-state preassembly facilitates rapid and specific G protein activation. However, no mechanism of preassembly has been described, and no functional consequences of preassembly have been demonstrated. Here we show that M(3) muscarinic acetylcholine receptors (M3R) form inactive-state complexes with G(q) heterotrimers in intact cells. The M3R C terminus is sufficient, and a six-amino-acid polybasic sequence distal to helix 8 ((565)KKKRRK(570)) is necessary for preassembly with G(q). Replacing this sequence with six alanine residues prevents preassembly, slows the rate of G(q) activation and decreases steady-state agonist sensitivity. That other G(q)-coupled receptors possess similar polybasic regions and also preassemble with G(q) suggests that these GPCRs may use a common preassembly mechanism to facilitate activation of G(q) heterotrimers.

Regulation of α(2B)-adrenergic receptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation by ADP-ribosylation factor 1.

A number of signaling molecules are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway by G protein-coupled receptors. In this study, we have demonstrated that α(2B)-adrenergic receptor (α(2B)-AR) interacts with ADP-ribosylation factor 1 (ARF1), a small GTPase involved in vesicle-mediated trafficking, in an agonist activation-dependent manner and that the interaction is mediated through a unique double Trp motif in the third intracellular loop of the receptor. Interestingly, mutation of the double Trp motif and siRNA-mediated depletion of ARF1 attenuate α(2B)-AR-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) without altering receptor intracellular trafficking, whereas expression of the constitutively active mutant ARF1Q71L and ARNO, a GDP-GTP exchange factor of ARF1, markedly enhances the activation of Raf1, MEK1, and ERK1/2. These data strongly demonstrate that the small GTPase ARF1 modulates ERK1/2 activation by α(2B)-AR and provide the first evidence indicating a novel function for ARF1 in regulating the MAPK signaling pathway.

Di-acidic motifs in the membrane-distal C termini modulate the transport of angiotensin II receptors from the endoplasmic reticulum to the cell surface.

The molecular mechanisms underlying the endoplasmic reticulum (ER) export and cell surface transport of nascent G protein-coupled receptors (GPCRs) have just begun to be revealed and previous studies have shown that hydrophobic motifs in the putative amphipathic 8(th) α-helical region within the membrane-proximal C termini play an important role. In this study, we demonstrate that di-acidic motifs in the membrane-distal, nonstructural C-terminal portions are required for the exit from the ER and transport to the plasma membrane of angiotensin II receptors, but not adrenergic receptors. More interestingly, distinct di-acidic motifs dictate optimal export trafficking of different angiotensin II receptors and export ability of each acidic residue in the di-acidic motifs cannot be fully substituted by other acidic residue. Moreover, the function of the di-acidic motifs is likely mediated through facilitating the recruitment of the receptors onto the ER-derived COPII transport vesicles. Therefore, the di-acidic motifs located in the membrane-distal C termini may represent the first linear motifs which recruit selective GPCRs onto the COPII vesicles to control their export from the ER.

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.

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.

Rab8 interacts with distinct motifs in alpha2B- and beta2-adrenergic receptors and differentially modulates their transport.

The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of alpha(2B)- and beta(2)-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of alpha(2B)-AR than beta(2)-AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by alpha(2B)-AR, but not beta(2)-AR, and arrested alpha(2B)-AR in the TGN compartment. Co-immunoprecipitation revealed that both alpha(2B)-AR and beta(2)-AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked beta(2)-AR interaction with Rab8, whereas mutation of residues Val(431)-Phe(432)-Asn(433)-Gln(434), Pro(447)-Trp(448), Gln(450)-Thr(451), and Trp(453) in the C terminus impaired alpha(2B)-AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric beta(2)-AR carrying the alpha(2B)-AR C terminus was similar to alpha(2B)-AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of alpha(2B)-AR and beta(2)-AR and differentially modulates their traffic from the TGN to the cell surface.

Deregulated Rac1 Activity in Neural Crest Controls Cell Proliferation, Migration and Differentiation During Midbrain Development.

Mutations in RAC1 allele are implicated in multiple brain tumors, indicating a rigorous control of Rac1 activity is required for neural tissue normal development and homeostasis. To understand how elevated Rac1 activity affects neural crest cells (NCCs) development, we have generated Rac1 CA ;Wnt1-Cre2 mice, in which a constitutively active Rac1 G12V mutant is expressed specifically in NCCs derivatives. Our results revealed that augmented Rac1 activity leads to enlarged midbrain and altered cell density, accompanied by increased NCCs proliferation rate and misrouted cell migration. Interestingly, our experimental data also showed that elevated Rac1 activity in NCCs disrupts regionalization of dopaminergic neuron progenitors in the ventral midbrain and impairs their differentiation. These findings shed light on the mechanisms of RAC1 mutation correlated brain tumor at the cellular and molecular level.

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.

In vitro and in vivo antitumor activity of melatonin receptor agonists.

Melatonin has been shown to inhibit the proliferation of estrogen receptor α (ERα)-positive human breast cancer cells in vitro and suppress the growth of carcinogen-induced mammary tumors in rats. Melatonin's antiproliferative effect is mediated, at least in part, through the MT1 melatonin receptor and mechanisms involving modulation of the estrogen-signaling pathway. To develop melatonin analogs with greater therapeutic effects, we have examined the in vitro and in vivo antimitotic activity of two MT1/MT2 melatonin receptor agonists, S23219-1 and S23478-1. In our studies, both agonists are quite effective at suppressing the growth of MCF-7 human breast cancer cells. At a concentration of 10⁻6 m, S23219-1 and S23478-1 inhibited the growth of MCF-7 cells by 60% and 73%, respectively. However, S23478-1 is more effective than melatonin and S23219-1 at repressing the expression and transactivation of the ERα, and modulating the expression of pancreatic spasmolytic polypeptide (pS2), an estrogen-regulated gene. The melatonin agonist S23478-1 exhibited enhanced antitumor potency in the subsequent studies in our animal model. At a dosage of 25 mg/kg/day, S23478-1 is more efficacious than melatonin at inducing regression of the established N-nitroso-N-methyl-urea-induced rat mammary tumors. This dose of S23478-1 (25 mg/kg/day) generated a significant (P < 0.05) overall regression response of 52%. Furthermore, at this dosage, S23478-1 is more effective than melatonin at suppressing the estrogen-signaling pathway and promoting tumor cell apoptosis, significantly increasing the expression of the pro-apoptotic protein Bax, while decreasing the expression of ERα and the anti-apoptotic protein Bcl-2.

Anterograde trafficking of G protein-coupled receptors: function of the C-terminal F(X)6LL motif in export from the endoplasmic reticulum.

We have reported previously that the F(X)(6)LL motif in the C termini is essential for export of alpha(2B)-adrenergic (alpha(2B)-AR) and angiotensin II type 1 receptors (AT1Rs) from the endoplasmic reticulum (ER). Here, we further demonstrate that mutation of the F(X)(6)LL motif similarly abolished the cell-surface expression of alpha(2B)-AR, AT1R, alpha(1B)-AR, and beta(2)-AR, suggesting that the F(X)(6)LL motif plays a general role in ER export of G protein-coupled receptors (GPCRs). Mutation of Phe to Val, Leu, Trp, and Tyr, and mutation of LL to FF and VV, markedly inhibited alpha(2B)-AR transport, indicating that the F(X)(6)LL function cannot be fully substituted by other hydrophobic residues. The structural analysis revealed that the Phe residue in the F(X)(6)LL motif is buried in the transmembrane domains and possibly interacts with Ile58 in beta(2)-AR and Val42 in alpha(2B)-AR, whereas the LL motif is exposed to the cytosolic space. Indeed, mutation of Ile58 in beta(2)-AR and Val42 in alpha(2B)-AR markedly disrupted cell surface transport of the receptors. It is noteworthy that the Val and Ile residues are highly conserved among the GPCRs carrying the F(X)(6)LL motif. Furthermore, the Phe mutant exhibited a stronger interaction with ER chaperones and was more potently rescued by physical and chemical treatments than the LL mutant. These data suggest that the Phe residue is probably involved in folding of alpha(2B)-AR and beta(2)-AR, possibly through interaction with other hydrophobic residues in neighboring domains. These data also provide the first evidence implying crucial roles of the C termini possibly through modulating multiple events in anterograde trafficking of GPCRs.

Alteration of the MT1 melatonin receptor gene and its expression in primary human breast tumors and breast cancer cell lines.

The MT1 melatonin receptor is bound and activated by the pineal hormone melatonin. This G protein-coupled melatonin receptor is expressed in human breast tumor cell lines, and when activated, mediates the growth-suppressive and steroid hormone/nuclear receptor modulatory actions of melatonin on breast tumor cells. In the current studies, we have examined the expression of the MT1 receptor in breast cancer cell lines and primary human breast tumors and correlated MT1 receptor expression with the deletion, rearrangement and amplification of the MT1 gene and established markers of breast cancer such as tumor size, stage, estrogen receptor alpha (ERalpha) and progesterone receptor (PR) expression. Theses studies suggest amplification of the MT1 gene in some breast tumors and an inverse correlation with ERalpha, PR and MT1 protein expression. Furthermore, these approaches employing immunohistochemical and immunofluorescent/confocal microscopic studies demonstrate that the MT1 receptor is localized to the caveoli and that MT1 expression in MCF-7 breast cancer cells can be repressed by estradiol and melatonin.

Endoplasmic reticulum export of adrenergic and angiotensin II receptors is differentially regulated by Sar1 GTPase.

The molecular mechanism underlying the export of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) remains largely unknown. In this manuscript, we investigated the role of Sar1 GTPase, which coordinates the assembly and budding of COPII-coated vesicles, in the cell-surface targeting, signaling and ER export of alpha(2B)-adrenergic (alpha(2B)-AR), beta(2)-AR and angiotensin II type 1 receptors (AT1R). The cell-surface expression of alpha(2B)-AR, beta(2)-AR and AT1R, and receptor-mediated ERK1/2 activation were significantly attenuated by the GTP-bound mutant Sar1H79G, suggesting that export from the ER of these receptors is mediated through the Sar1-dependent COPII-coated vesicles. Interestingly, subcellular distribution analyses showed that alpha(2B)-AR and AT1R were highly concentrated at discrete locations near the nucleus in cells expressing Sar1H79G, whereas beta(2)-AR exhibited an ER distribution. These data indicate that Sar1-catalyzed efficient GTP hydrolysis differentially regulates ER export of adrenergic and angiotensin II receptors. These data provide the first evidence indicating distinct mechanisms for the recruitment of different GPCRs into the COPII vesicles on the ER membrane.

Expression pattern of Kmt2d in murine craniofacial tissues.

Formation of the calvaria is a multi-staged process and is regulated by multiple genetic factors. Disruption of normal calvarial development usually causes craniosynostosis, a prevalent birth defect characterized by premature fusion of calvarial bone. Recent studies have identified mutations of KMT2D allele in patients with craniosynostosis, indicating a potential role for Kmt2d in calvarial development. KMT2D mutations have also been implicated in Kabuki syndrome, which features a distinct facial appearance, skeletal abnormality, growth retardation and intellectual disability. However, the expression pattern of Kmt2d has not been fully elucidated. In the present study we examined the expression pattern of Kmt2d at multiple stages of embryo development in mice, with a focus on the craniofacial tissues. Our in situ hybridization results showed that Kmt2d mRNA is expressed in the developing calvarial osteoblasts, epithelia and neural tissues. Such an expression pattern is in line with the phenotypes of Kabuki syndrome, suggesting that Kmt2d plays an intrinsic role in normal development and homeostasis of these craniofacial tissues.

Regulation of G protein-coupled receptor export trafficking.

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.