Elsevier

Brain Research

Volume 780, Issue 2, 12 January 1998, Pages 311-322
Brain Research

Research report
Trafficking itineraries of G protein-coupled receptors in epithelial cells do not predict receptor localization in neurons

https://doi.org/10.1016/S0006-8993(97)01216-XGet rights and content

Abstract

These studies explored whether the localization and differential trafficking itineraries of G protein-coupled receptors in polarized renal epithelial cells might predict their localization in neurons, as suggested previously. The A1 adenosine receptor is preferentially localized apically, whereas the three α2-adrenergic receptor (α2AR) subtypes are localized on the lateral subdomain of Madin–Darby canine kidney cells. The α2A AR and α2C AR subtypes achieve this localization by direct targeting; α2B AR is randomly delivered but preferentially retained basolaterally. Despite their differing itineraries in renal epithelial cells, all three epitope-tagged α2-adrenergic receptor subtypes were found in neuronal cell bodies and along the entire length of the neuronal processes following transfection into long term primary cultures of mouse embryonic spinal cord neurons. In a small fraction of neuronal cells, expression of A1 adenosine receptor was limited to a short segment of their processes, otherwise it too was distributed in the soma and neuronal processes. A mutant α2A AR that exhibits an accelerated turnover on the surface of epithelial cells nonetheless has a localization pattern indistinguishable from the wild-type α2A AR in spinal cord neurons. Thus, unlike examples for GPI-anchored apical proteins that distribute along axons or single transmembrane-spanning basolateral proteins that localize to the soma of neurons, similar predictions do not appear to apply for polytopic G protein-coupled receptors.

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

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    Current address, Department of Medicine, Division of Nephrology, Washington University School of Medicine, St. Louis, MO.

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