Research reportTrafficking itineraries of G protein-coupled receptors in epithelial cells do not predict receptor localization in neurons
Introduction
Epinephrine and norepinephrine mediate diverse physiological functions via interaction with α2-adrenergic receptors (α2-ARs), including regulation of transepithelial sodium and water transport and suppression of neurotransmitter release in the central and peripheral nervous system. Localization of receptor molecules on the appropriate surface is crucial for receptor-mediated vectorial flow of regulatory information in these polarized cell systems, and may contribute as well to a higher order of specificity of receptor coupling to particular G proteins and effectors based in their proximity in a given surface microdomain.
Previously, we characterized the steady-state localization and trafficking itineraries of all three α2-AR subtypes in polarized Madin–Darby canine kidney (MDCKII) cells. As predicted from functions in vivo 14, 24, all three α2-AR subtypes are localized to the basolateral surface of MDCKII cells at steady-state. Moreover, immunofluorescence studies have suggested that all three α2-AR subtypes are enriched on the lateral subdomain of epithelial cells [32]. The α2A-AR and α2C-AR subtypes achieve this basolateral localization via direct delivery, as revealed in pulse-chase studies, whereas the α2B-AR appears to be randomly delivered to both surfaces, but rapidly leaves the apical surface (t1/2=15–30 min), resulting in enrichment on the basolateral surface at steady-state (t1/2=8–10 h) [32]. The basolateral delivery of the α2A-AR subtype does not appear to rely on functional coupling to the Gi/Go subpopulation of G proteins that mediates their physiological effects at the cell surface, since pertussis toxin treatment does not alter the targeted delivery of the α2A-AR to the basolateral surface [11]. The α2C-AR, in contrast to the α2A-AR and α2B-AR subtypes, is distributed at steady-state not only on the cell surface but also in an intracellular compartment distinct from endosomes [28]but not yet fully characterized [32]. Interestingly, the adenosine type-1 receptor (A1AdoR), which also couples to the Gi/Go subpopulation of G proteins, is enriched (65–70%) on the apical surface of MDCKII and LLCPK1 cells at steady-state, achieved by direct delivery to that surface [21].
Since all three α2-AR subtypes are localized in the CNS 5, 26, and α2-AR agonists suppress neurotransmitter release in the central and peripheral nervous system, it was of interest to determine whether the differential trafficking itineraries of the α2-AR subtypes in renal epithelial cells might predict their localization in neurons. Effects of α2-AR agonists on neuronal ion currents and neurotransmitter release have been measured both on the somatodendritic membrane and in axonal terminals 1, 4, 18, 22. Early comparisons of protein localization in epithelial versus neuronal cells suggest that apically-targeted molecules, such as GPI-anchored proteins, appeared along axons in neurons, whereas basolaterally-targeted molecules, such as vesicular stomatitis virus (VSV) glycoprotein, were detected on the somatodendritic membrane of neurons 2, 3. However, further studies revealed that exceptions to this paradigm exist [17].
To evaluate whether the differing trafficking itineraries of the three α2-AR subtypes or the opposite polarization of α2-AR subtypes (basolateral) and the A1AdoR (apical) in epithelial cells is paralleled by distinctive neuronal localization, we examined the distribution of epitope-tagged versions of these molecules following their introduction into well-established primary cultures of mouse embryonic spinal cord neurons. Since deletion of the predicted large third intracellular loop of the α2A-AR results in acceleration of its turnover on the basolateral surface of MDCKII cells [12], perhaps due to lack of interaction of this mutant structure with presumptive tethering protein, we also evaluated whether this mutant α2A-AR structure manifested a different distribution in neuronal cells when compared to the wild-type α2A-AR.
Section snippets
Materials
The protein A-purified 12CA5 monoclonal antibody was purchased from the Berkley Antibody (BABCO). Mouse monoclonal anti-MAP2 and anti-tau antibodies were obtained from Boehringer-Mannheim Biochemicals. Rabbit polyclonal anti-MAP and anti-tau antibodies were from Sigma. Cy3-conjugated donkey anti-mouse IgG, Cy3-conjugated goat anti-rabbit, FITC-conjugated donkey anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG were purchased from Jackson Immunochemicals. Supplemented Minimal Essential
Results
We utilized primary neuronal cell cultures derived from spinal cords of 13-day old mouse embryos that have been characterized both electrophysiologically and pharmacologically 29, 30, 31as recipients for cDNAs expressing the receptor of interest. Previous studies also have documented that the expression of various neuronal markers in cultures of mouse embryonic spinal cord reveals a well polarized neuronal phenotype, permitting a reliable morphological characterization of heterogeneously
Distribution of the α2-AR subtypes in cultured spinal cord neurons
Functional significance of the precise expression of α2-ARs in neuronal cells has been widely recognized. Previous reports demonstrate that α2-ARs present in specific regions of the CNS mediate various physiological responses. For example, α2-ARs in locus ceruleus are involved in pain perception and anaesthetic sparing responses [6]; α2-ARs in the ventral lateral medulla mediate central control of blood pressure [20]. Vectorial transfer of information via α2-AR in the CNS potentially can be
Acknowledgements
We are grateful to Dr. Gary A. Banker (University of Virginia) for his insights concerning receptor and marker protein localization in cultured neurons, and to Dr. Michael McLean and Ronald Thomas (Vanderbilt University, Department of Neurology) for their invaluable advice on the isolation and maintenance of cultured primary embryonic spinal cord neurons. We also wish to thank Dr. Ford Ebner (Vanderbilt University, Department of Cell Biology) for his generous gift of anti-tau and anti-MAP2
References (32)
- et al.
Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture
Cell
(1990) - et al.
Sorting mechanisms of tau and MAP2 in neurons: suppressed axonal transit of MAP2 and locally regulated microtubule binding
Neuron
(1995) - et al.
The α2A-adrenergic receptor is targeted directly to the basolateral membrane domain of Madin-Darby canine kidney cells independent of coupling to pertussis toxin-sensitive GTP-binding proteins
J. Biol. Chem.
(1993) - et al.
Peripheral nociceptive effects of α2-adrenergic receptor agonists in the rat
Neuroscience
(1995) - et al.
Distribution of α1- and α2-adrenoceptors in brush border and basolateral membrane from rat kidney cortical tubules
Jpn. J. Pharmacol.
(1987) - et al.
Receptors coupled to pertussis toxin-sensitive G-proteins traffic to opposite surfaces in Madin-Darby canine kidney cells
J. Biol. Chem.
(1996) - et al.
Presynaptic opioid receptor and α2-adrenoceptor-mediated inhibition of noradrenaline release in the rat brain: Role of hyperpolarization?
Eur. J. Pharmacol.
(1984) - et al.
Getting the message from the gene to the synapse: sorting and intracellular transport of RNA in neurons
TINS
(1992) - et al.
Noradrenergic and opioid systems interact to alter the detection of noxious thermal stimuli and facial scratching in monkeys
Pain
(1993) - et al.
Subtype specific differences in the intracellular sorting of G protein-coupled receptors
J. Biol. Chem.
(1993)
The three α2-adrenergic receptor subtypes achieve basolateral localization in Madin-Darby canine kidney II cells via different targeting mechanisms
J. Biol. Chem.
α2-Adrenoceptor-mediated hyperpolarization of locus ceruleus neurons: Intracellular studies in vivo
Science
Polarized sorting of glypiated proteins in hippocampal neurons
Nature
Norepinephrine-mediated synaptic inhibition in rat locus ceruleus neurons
J. Physiol.
α2-adrenoceptor subtypes identified by [3H]-RX821002 binding in the human brain: the agonist guanoxabenz does not discriminate different forms of the predominant alpha-2 subtype
J. Neurochem.
Dexmedetomidine injection into the locus ceruleus produces antinociception
Anesthesiology
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2004, Journal of Biological ChemistryCitation Excerpt :In contrast, sorting in epithelial cells may be less conclusive because it relies on an extrapolation from the approximative rule that apical sorting in polarized epithelial cells predicts axonal targeting in neuronal cells. In fact, there is precedence for transmembrane proteins that do not conform to this rule (24, 25). Finally we do not consider that our analysis is confounded by the presence of a fluorescent tag at the amino terminus of GAT1 because YFP-GAT1 was delivered to the correct membrane compartment, that is the plasma membrane of the neurite extension of differentiated PC12 cells as well as to the axonal compartment of hippocampal neurons.
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Current address, Department of Medicine, Division of Nephrology, Washington University School of Medicine, St. Louis, MO.