Carl M. Hurt

High constitutive activity of the human formyl peptide receptor.

The formyl peptide receptor (FPR) couples to pertussis toxin (PTX)-sensitive Gi-proteins to activate chemotaxis and exocytosis in neutrophils. PTX reduces not only formyl peptide-stimulated but also agonist-independent (“basal”) Gi-protein activity, suggesting that the FPR is constitutively active. We aimed at identifying an inverse FPR agonist,i.e. a compound that suppresses constitutive FPR activity. In Sf9 insect cell membranes, the G-protein heterotrimer Giα2β1γ2reconstitutedN-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-stimulated guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding and GTPγS-sensitive high affinity [3H]FMLP binding. The FPR “antagonist” cyclosporin H (CsH) potently and efficiently reduced basal GTPγS binding in Sf9 membranes. Another FPR antagonist,N-t-butoxycarbonyl-l-phenylalanyl-l-leucyl-l-phenylalanyl-l-leucyl-l-phenylalanine did not inhibit basal GTPγS binding but blocked the inhibitory effect of CsH on GTPγS binding. Na+ reduced basal GTPγS binding and eliminated the inhibitory effect of CsH. Similar effects of FMLP, CsH, and Na+ as in Sf9 membranes were observed with FPR expressed in the mammalian cell line HEK293. Our data show that the human FPR possesses high constitutive activity. CsH is an inverse FPR agonist and stabilizes the FPR in an inactive state. Na+ also stabilizes the FPR in an inactive state and, thereby, diminishes inverse agonist efficacy.

Organization of β-adrenoceptor signaling compartments by sympathetic innervation of cardiac myocytes

The sympathetic nervous system regulates cardiac function through the activation of adrenergic receptors (ARs). β1 and β2ARs are the primary sympathetic receptors in the heart and play different roles in regulating cardiac contractile function and remodeling in response to injury. In this study, we examine the targeting and trafficking of β1 and β2ARs at cardiac sympathetic synapses in vitro. Sympathetic neurons form functional synapses with neonatal cardiac myocytes in culture. The myocyte membrane develops into specialized zones that surround contacting axons and contain accumulations of the scaffold proteins SAP97 and AKAP79/150 but are deficient in caveolin-3. The β1ARs are enriched within these zones, whereas β2ARs are excluded from them after stimulation of neuronal activity. The results indicate that specialized signaling domains are organized in cardiac myocytes at sites of contact with sympathetic neurons and that these domains are likely to play a role in the subtype-specific regulation of cardiac function by β1 and β2ARs in vivo.

Cell-type Specific Targeting of the α2c-Adrenoceptor EVIDENCE FOR THE ORGANIZATION OF RECEPTOR MICRODOMAINS DURING NEURONAL DIFFERENTIATION OF PC12 CELLS

We have previously shown differences in the intracellular targeting of α2a (α2A)- and α2c (α2C)-adrenoreceptors expressed in the same cell line (von Zastrow, M., Link, R., Daunt, D., Barsh, G., and Kobilka, B. (1993) J. Biol. Chem. 268, 763–766; Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711–720). α2A-Adrenoreceptors reside primarily in the plasma membrane in HEK 293 cells, while co-expressed α2C-adrenoreceptors are found mainly in an intracellular compartment. Since α2c-adrenoreceptors are expressed primarily in the brain, we compared the intracellular targeting of α2C-adrenoreceptors in two neuroendocrine cell lines with the targeting in three epithelial cell lines and one fibroblast cell line. In transiently transfected COS7 cells, and in stably transfected normal rat kidney cells, Madin-Darby canine kidney cells, and Rat1 fibroblasts, a significant proportion of α2C-adrenoreceptor detected by immunocytochemistry co-localized with markers for both the endoplasmic reticulum and the cis/medial Golgi compartments. In contrast, both PC12 cells and AtT20 cells efficiently targeted α2C-adrenoreceptors to the plasma membrane. Ligand binding and Western blot analyses indicate that intracellular receptor in normal rat kidney cells is functional and undergoes normal post-translational processing. In PC12 cells the expressed α2C-adrenoreceptors become concentrated in neurite outgrowths in discrete regions of the plasma membrane having a high density of F-actin following treatment with nerve growth factor. These findings provide evidence for cell-type specific factors that facilitate the targeting of the G protein-coupled receptors to the plasma membrane.

Regulation of G-Protein Coupled Receptor Traffic by an Evolutionary Conserved Hydrophobic Signal

Plasma membrane (PM) expression of G‐protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c‐adrenergic receptors (α2c‐ARs), heterologous expression in non‐native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric α2a/α2c‐AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for α2c‐AR subtype‐specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into α2a‐ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within α2c‐ARs serves as a regulator of GPCR trafficking.

Differential targeting and function of α2A and α2C adrenergic receptor subtypes in cultured sympathetic neurons

Previous research suggested that alpha2A and alpha2C adrenergic receptor (AR) subtypes have overlapping but unique physiological roles in neuronal signaling; however, the basis for these dissimilarities is not completely known. To better understand the observed functional differences between these autoreceptors, we investigated targeting and signaling of endogenously expressed alpha2A and alpha2CARs in cultured sympathetic ganglion neurons (SGN). At Days 1 and 4, alpha2A and alpha2CARs could be readily detected in SGN from wild-type mice. By Day 8, alpha2A ARs were targeted to cell body, as well as axonal and dendritic sites, whereas alpha2C ARs were primarily localized to an intracellular vesicular pool within the cell body and proximal dendritic projections. Expression of synaptic vesicle marker protein SV2 did not differ at Day 8 nor co-localize with either subtype. By Day 16, however, alpha2C ARs had relocated to somatodendritic and axonal sites and, unlike alpha2A ARs, co-localized with SV2 at synaptic contact sites. Consistent with a functional role for alpha2 ARs, we also observed that dexmedetomidine stimulation of cultured SGN more efficiently inhibited depolarization-induced calcium entry into older, compared to younger, cultures. These results provide direct evidence of distinct developmental patterns of endogenous alpha2A and alpha2C AR targeting and function in a native cell system and that maturation of SGN in culture leads to alterations in neuronal properties required for proper targeting. More importantly, the co-localization at Day 16 of alpha2C ARs at sites of synaptic contact may partially explain the differential modulation of neurotransmitter release and responsiveness to action potential frequency observed between alpha2A and alpha2C ARs in SGN.

REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins. Therefore, some REEPs can be further described as ER membrane shaping adapter proteins.

Systematic and Quantitative Analysis of G Protein-Coupled Receptor Trafficking Motifs

Plasma membrane expression of G protein-coupled receptors (GPCRs) is a dynamic process balancing anterograde and retrograde trafficking. Multiple interrelated cellular processes determine the final level of cell surface expression, including endoplasmic reticulum (ER) export/retention, receptor internalization, recycling, and degradation. These processes are highly regulated to achieve specific localization to subcellular domains (e.g., dendrites or basolateral membranes) and to affect receptor signaling. Analysis of potential ER trafficking motifs within GPCRs requires careful consideration of intracellular dynamics, such as protein folding, ER export and retention, and glycosylation. This chapter presents an approach and methods for qualitative and quantitative assessment of these processes to aid in accurate identification of GPCR trafficking motifs, utilizing the analysis of a hydrophobic extracellular trafficking motif in α2C adrenergic receptors as a model system.

REEP1 and REEP2 proteins are preferentially expressed in neuronal and neuronal-like exocytotic tissues

The six members of the Receptor Expression Enhancing Protein (REEP) family were originally identified based on their ability to enhance heterologous expression of olfactory receptors and other difficult to express G protein-coupled receptors. Interestingly, REEP1 mutations have been linked to neurodegenerative disorders of upper and lower motor neurons, hereditary spastic paraplegia (HSP) and distal hereditary motor neuropathy type V (dHMN-V). The closely related REEP2 isoform has not demonstrated any such disease linkage. Previous research has suggested that REEP1 mRNA is ubiquitously expressed in brain, muscle, endocrine, and multiple other organs, inconsistent with the neurodegenerative phenotype observed in HSP and dHMN-V. To more fully examine REEP1 expression, we developed and characterized a new REEP1 monoclonal antibody for both immunoblotting and immunofluorescent microscopic analysis. Unlike previous RT-PCR studies, immunoblotting demonstrated that REEP1 protein was not ubiquitous; its expression was restricted to neuronal tissues (brain, spinal cord) and testes. Gene expression microarray analysis demonstrated REEP1 and REEP2 mRNA expression in superior cervical and stellate sympathetic ganglia tissue. Furthermore, expression of endogenous REEP1 was confirmed in cultured murine sympathetic ganglion neurons by RT-PCR and immunofluorescent staining, with expression occurring between Day 4 and Day 8 of culture. Lastly, we demonstrated that REEP2 protein expression was also restricted to neuronal tissues (brain and spinal cord) and tissues that exhibit neuronal-like exocytosis (testes, pituitary, and adrenal gland). In addition to sensory tissues, expression of the REEP1/REEP2 subfamily appears to be restricted to neuronal and neuronal-like exocytotic tissues, consistent with neuronally restricted symptoms of REEP1 genetic disorders.

Transient Receptor Potential Ankyrin 1 Activation within the Cardiac Myocyte Limits Ischemia–reperfusion Injury in Rodents

Background:Recent evidence suggests that cross talk exists between cellular pathways important for pain signaling and ischemia–reperfusion injury. Here, the authors address whether the transient receptor potential ankyrin 1 (TRPA1) channel, important in pain signaling, is present in cardiac myocytes and regulates cardiac ischemia–reperfusion injury. Methods:For biochemical analysis of TRPA1, techniques including quantitative polymerase chain reaction, Western blot, and immunofluorescence were used. To determine how TRPA1 mediates cellular injury, the authors used an in vivo model of rat cardiac ischemia–reperfusion injury and adult rat–isolated cardiac myocytes subjected to hypoxia–reoxygenation. Results:The authors’ biochemical analysis indicates that TRPA1 is within the cardiac myocytes. Further, using a rat in vivo model of cardiac injury, the TRPA1 activators ASP 7663 and optovin reduce myocardial injury (45 ± 5%* and 44 ± 8%,* respectively, vs. control, 66 ± 6% infarct size/area at risk; n = 6 per group; mean ± SD; *P < 0.001). TRPA1 inhibition also blocked the infarct size–sparing effects of morphine. In isolated cardiac myocytes, the TRPA1 activators ASP 7663 and optovin reduce cardiac myocyte cell death when given during reoxygenation (20 ± 3%* and 22 ± 4%* vs. 36 ± 3%; percentage of dead cells per field, n = 6 per group; mean ± SD; *P < 0.05). For a rat in vivo model of cardiac injury, the infarct size–sparing effect of TRPA1 activators also occurs during reperfusion. Conclusions:The authors’ data suggest that TRPA1 is present within the cardiac myocytes and is important in regulating myocardial reperfusion injury. The presence of TRPA1 within the cardiac myocytes may potentially explain why certain pain relievers that can block TRPA1 activation, such as cyclooxygenase-2 inhibitors or some nonsteroidal antiinflammatory drugs, could be associated with cardiovascular risk.

Common α2A and α2C adrenergic receptor polymorphisms do not affect plasma membrane trafficking

Various naturally occurring polymorphic forms of human G protein-coupled receptors (GPCRs) have been identified and linked to diverse pathological diseases, including receptors for vasopressin type 2 (nephrogenic diabetes insipidus) and gonadotropin releasing hormone (hypogonadotropic hypogonadism). In most cases, polymorphic amino acid mutations disrupt protein folding, altering receptor function as well as plasma membrane expression. Other pathological GPCR variants have been found that do not alter receptor function, but instead affect only plasma membrane trafficking (e.g., delta opiate and histamine type 1 receptors). Thus, altered membrane trafficking with retained receptor function may be another mechanism causing polymorphic GPCR dysfunction. Two common human α2A and α2C adrenergic receptor (AR) variants have been identified (α2A N251K and α2C Δ322-325 ARs), but pharmacological analysis of ligand binding and second messenger signaling has not consistently demonstrated altered receptor function. However, possible alterations in plasma membrane trafficking have not been investigated. We utilized a systematic approach previously developed for the study of GPCR trafficking motifs and accessory proteins to assess whether these α2 AR variants affected intracellular trafficking or plasma membrane expression. By combining immunofluorescent microscopy, glycosidic processing analysis, and quantitative fluorescent-activated cell sorting (FACS), we demonstrate that neither variant receptor had altered intracellular localization, glycosylation, nor plasma membrane expression compared to wild-type α2 ARs. Therefore, pathopharmacological properties of α2A N251K and α2C Δ322-325 ARs do not appear to be due to altered receptor pharmacology or plasma membrane trafficking, but may involve interactions with other intracellular signaling cascades or proteins.