Intracellular trafficking of a tagged and functional mammalian olfactory receptor (original) (raw)
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Journal of Biological Chemistry, 2001
The cGMP-dependent protein kinases (PKGs) are ubiquitous effector enzymes that regulate a variety of physiological processes in response to nitric oxide and natriuretic agonists. We have constructed green fluorescent fusion proteins (GFP) using full-length (PKG-GFP) and truncations encoding either the regulatory domain of PKG1␣ (G1␣R-GFP) or the catalytic domains of PKG1␣ (GFP-G1C) to examine the enzymatic properties and intracellular location. When transiently transfected into mammalian cells, these constructs were detected on Western blots at the expected sizes using anti-GFP antibodies. The GFP-G1C and the full-length PKG1␣-GFP fusion proteins were found to have constitutive activity both in vivo and in vitro. The G1␣R-GFP protein was found to dimerize with endogenous type 1 PKG and behaved in a dominant negative manner both in vivo and in vitro. When expressed transiently in either HEK-293 or A549 epithelial cells, the fusion proteins encoding the amino-terminal regulatory domains (PKG-GFP, G1␣R-GFP) were present in the cytosol and were rarely observed in the nucleus. In contrast, the GFP-G1C (lacking regulatory domains) concentrated in the nucleus. Of the fusion proteins containing the regulatory region, the constitutive PKG-GFP protein was present in a more centralized location, whereas the G1␣R-GFP protein colocalized with F-actin on stress fibers and in dynamic regions of the plasma membrane. Microscopic and immunoprecipitation studies indicated that both the G1␣R-GFP and the PKG-GFP fusion proteins colocalized with vasodilator-stimulated phosphoprotein (VASP). These constructs thus represent novel tools with which to visualize inactive, and activated, PKG1␣ in vivo, and we have used them to demonstrate two functionally independent domains. In addition, we show for the first time in living cells that PKG is found in dynamic membrane regions in association with VASP.
Electrophysiological and Fluorescence Microscopy Studies with HERG Channel/EGFP Fusion Proteins
Journal of Membrane Biology, 2008
HERG (human ether-a-go-go-related gene) encodes the Kv11.1 protein α-subunit that underlies the rapidly activating delayed rectifier K+ current (I Kr) in the heart. Alterations in the functional properties or membrane incorporation of HERG channels, either by genetic mutations or by administration of drugs, play major roles in the development of life-threatening torsades de pointes cardiac arrhythmias. Visualization of ion channel localization is facilitated by enhanced green fluorescent protein (EGFP) tagging, but this process can alter their properties. The aim of the present study was to characterize the electrophysiological properties and the cellular localization of HERG channels in which EGFP was tagged either to the C terminus (HERG/EGFP) or to the N terminus (EGFP/HERG). These fusion constructs were transiently expressed in human embryonic kidney (HEK) 293 cells, and the whole-cell patch-clamp configuration and a confocal laser scanning microscope with primary anti-HERG antibodies and fluorescently labeled secondary antibodies were used. For EGFP/HERG channels the deactivation kinetics were faster and the peak tail current density was reduced when compared to both wild-type HERG channels and HERG/EGFP channels. Laser scanning microscopic studies showed that both fusion proteins were localized in the cytoplasm and on discrete microdomains in the plasma membrane. The extent of labeling with anti-HERG antibodies of HEK 293 cells expressing EGFP/HERG channels was less when compared to HERG/EGFP channels. In conclusion, both electrophysiological and immunocytochemical studies showed that EGFP/HERG channels themselves have a protein trafficking defect. HERG/EGFP channels have similar properties as untagged HERG channels and, thus, might be especially useful for fluorescence microscopy studies.
Cellular signaling by an agonist-activated receptor/Gs alpha fusion protein
Proceedings of the National Academy of Sciences of the United States of America, 1994
The consequences of agonist-dependent activation of guanine nucleotide-binding protein (G protein)-coupled receptors vary from cell to cell, depending on a complex network of regulations between components of the signaling cascade. Specific interactions between receptors, G proteins, and effectors are difficult to analyze in intact cells. Engineering of receptor-transducer fusion proteins might be an effective strategy to target cellular effectors more efficiently and specifically. As a model, we evaluated the ability of a fusion protein of beta 2-adrenergic receptor bound to the alpha subunit of adenylyl cyclase-stimulatory G protein (Gs alpha) to restore the defective activation of adenylyl cyclase in S49 cyc- cells that lack endogenous Gs alpha. The coupling between the two partners of the fusion protein was functional, and the agonist-dependent activation of the effector was more potent and more productive in transfected than in wild-type S49 cells. The covalent link between rec...
Role of a Ubiquitously Expressed Receptor in the Vertebrate Olfactory System
Journal of Neuroscience, 2013
Odorant cues are recognized by receptors expressed on olfactory sensory neurons, the primary sensory neurons of the olfactory epithelium. Odorant receptors typically obey the "one receptor, one neuron" rule, in which the receptive field of the olfactory neuron is determined by the singular odorant receptor that it expresses. Odor-evoked receptor activity across the population of olfactory neurons is then interpreted by the brain to identify the molecular nature of the odorant stimulus. In the present study, we characterized the properties of a C family G-protein-coupled receptor that, unlike most other odorant receptors, is expressed in a large population of microvillous sensory neurons in the zebrafish olfactory epithelium and the mouse vomeronasal organ. We found that this receptor, OlfCc1 in zebrafish and its murine ortholog Vmn2r1, is a calcium-dependent, low-sensitivity receptor specific for the hydrophobic amino acids isoleucine, leucine, and valine. Loss-of-function experiments in zebrafish embryos demonstrate that OlfCc1 is required for olfactory responses to a diverse mixture of polar, nonpolar, acidic, and basic amino acids. OlfCc1 was also found to promote localization of other OlfC receptor family members to the plasma membrane in heterologous cells. Together, these results suggest that the broadly expressed OlfCc1 is required for amino acid detection by the olfactory system and suggest that it plays a role in the function and/or intracellular trafficking of other olfactory and vomeronasal receptors with which it is coexpressed.
G-protein βγ Subunit Genes Expressed in Olfactory Receptor Neurons
Chemical Senses, 1997
The expression of genes encoding G-protein 0y subunits was investigated in isolated olfactory receptor neurons from channel catfish. DNA sequencing of PCR products showed that the p1, P2, y2 and y3 genes were expressed in the neurons. Western blotting showed that at least three of these subunit proteins were expressed. This first analysis of the expression of Py genes in olfactory receptor neurons suggests that these subunits may be involved in a variety of transduction events in these cells.
A novel 45 kDa secretory protein from rat olfactory epithelium: primary structure and localisation
Febs Letters, 1999
cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46 026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.
Specificity of Olfactory Receptor Interactions with Other G Protein-coupled Receptors
Journal of Biological Chemistry, 2007
Studies on olfactory receptor (OR) pharmacology have been hindered by the poor plasma membrane localization of most ORs in heterologous cells. We previously reported that association with the  2-adrenergic receptor ( 2-AR) facilitates functional expression of the OR M71 at the plasma membrane of HEK-293 cells. In the present study, we examined the specificity of M71 interactions with other G protein-coupled receptors (GPCRs). M71 was co-expressed in HEK-293 cells with 42 distinct GPCRs, and the vast majority of these receptors had no significant effect on M71 surface expression. However, co-expression with three subtypes of purinergic receptor (P2Y 1 R, P2Y 2 R, and A 2A R) resulted in markedly enhanced plasma membrane localization of M71. Agonist stimulation of M71 co-expressed with P2Y 1 R and P2Y 2 R activated the mitogen-activated protein kinase pathway via coupling of M71 to G␣ o. We also examined the ability of  2-AR, P2Y 1 R, P2Y 2 R, and A 2A R to interact with and regulate ORs beyond M71. We found that coexpression of  2-AR or the purinergic receptors enhanced the surface expression for an M71 subfamily member but not for several other ORs from different subfamilies. In addition, through chimeric receptor studies, we determined that the second transmembrane domain of  2-AR is necessary for  2-AR facilitation of M71 plasma membrane localization. These studies shed light on the specificity of OR interactions with other GPCRs and the mechanisms governing olfactory receptor trafficking. Mammalian olfaction begins at the plasma membrane of olfactory sensory neuron (OSN) 2 cilia, where inhaled environmental chemicals bind and activate a subset of G protein-coupled receptors (GPCRs), termed olfactory receptors (ORs). Although ORs were identified over a decade ago and are encoded by the most numerous multigene family in mammals, remarkably few OR-ligand pairs have been characterized (1, 2).
Transduction Mechanisms in Vertebrate Olfactory Receptor Cells
Physiological Reviews, 1998
Schild, Detlev, and Diego Restrepo. Transduction Mechanisms in Vertebrate Olfactory Receptor Cells. Physiol. Rev. 78: 429–466, 1998. — Considerable progress has been made in the understanding of transduction mechanisms in olfactory receptor neurons (ORNs) over the last decade. Odorants pass through a mucus interface before binding to odorant receptors (ORs). The molecular structure of many ORs is now known. They belong to the large class of G protein-coupled receptors with seven transmembrane domains. Binding of an odorant to an OR triggers the activation of second messenger cascades. One second messenger pathway in particular has been extensively studied; the receptor activates, via the G protein Golf, an adenylyl cyclase, resulting in an increase in adenosine 3′,5′-cyclic monophosphate (cAMP), which elicits opening of cation channels directly gated by cAMP. Under physiological conditions, Ca2+has the highest permeability through this channel, and the increase in intracellular Ca2+...
2009
Background: Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. Methodology/Principal Findings: We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-b-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. Conclusions/Significance: We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons.
Molecular Brain Research, 1997
Replacing the G-protein-coupling domains of the b-adrenergic receptor with homologous domains of putative olfactory receptors 2 produced chimeric receptors which were able to stimulate pigment dispersion in Xenopus melanophores, a G-protein-mediated pathway. A multiple replacement chimera containing the second, third and C-terminal cytoplasmic domains of receptor OR5 elevated cyclic X X Ž. adenosine 3 :5-monophosphate cAMP and suppressed production of inositol phosphates. Co-expression of Ga did not alter the olf Ž. strength of response of this chimera. A novel rat olfactory receptor cDNA U131 was isolated and sequenced. Expression of U131 and OR5 constructs containing an N-terminal epitope-tag or C-terminal fusion to green fluorescent protein occurred in an intracellular network but not in the plasma membrane of heterologous cells. Similarly treated b-adrenergic receptors were functional and were 2 observed in the plasma membrane and the intracellular network. These results demonstrate that the putative cytoplasmic domains of olfactory receptors are capable of functional interaction with heterologous G-proteins of the Ga subtype. Instead, the absence of these s receptors from the plasma membrane of heterologous cells appears to explain our inability to determine if odorants can activate the olfactory receptor clones. We hypothesize that the olfactory receptors have requirements for maturation and targeting to the plasma membrane that are different from most other G-protein-coupled receptors. q 1997 Elsevier Science B.V.