EphB Receptors Co-Distribute with a Nicotinic Receptor Subtype and Regulate Nicotinic Downstream Signaling in Neurons

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Abstract

Activation of nicotinic acetylcholine receptors (nAChRs) on neurons engages calcium-dependent signaling pathways regulating numerous events. Receptors containing α7 subunits (α7-nAChRs) are prominent in this because of their abundance and high relative calcium permeability. We show here that EphB2 receptors are co-localized with postsynaptic α7-nAChRs on chick ciliary ganglion neurons and that treatment of the cells with an ephrinB1 construct to activate the EphB receptors exerts physical restraints on both classes of receptors, diminishing their dispersal after spine retraction or lipid raft disruption. Moreover, the ephrinB1/EphB receptor complex specifically enhances the ability of α7-nAChRs to activate the transcription factor CREB, acting through a pathway including a receptor tyrosine kinase, a Src family member, PI3 kinase, and protein kinase A most distally. The enhancement does not appear to result from a change in the α7-nAChR current amplitude, suggesting a downstream target. The results demonstrate a role for ephrin/EphB action in nicotinic signaling.

Introduction

EphB receptor tyrosine kinases are widely expressed in the brain (Yamaguchi and Pasquale, 2004). Activation of postsynaptic EphB2 receptors (EphB2Rs) by their presynaptic ligand ephrinB helps organize glutamatergic synapses. The extracellular portion of EphB2Rs interacts directly with NMDA receptors, promoting receptor clustering and synapse formation/maturation (Dalva et al., 2000). The intracellular portion of EphB2Rs is instrumental in augmenting calcium signaling by NMDA receptors and downstream effects, including altered gene expression (Takasu et al., 2002). Moreover, ephrinB/EphB2R interactions can recruit AMPA receptors to synaptic locations and support dendritic spine formation (Kayser et al., 2006).

A role for EphB2Rs in nicotinic signaling pathways has not been reported. The chick parasympathetic ciliary ganglion (CG) offers a useful preparation for examining the possibility. The CG contains ciliary neurons that innervate striated muscle in the iris and ciliary body, and choroid neurons that innervate smooth muscle in the choroid layer (Dryer, 1994). Both neuronal populations receive cholinergic input from the accessory oculomotor nucleus in the midbrain which activates homopentameric α7-containing nicotinic acetylcholine receptors (α7-nAChRs) and heteromeric α3*-nAChRs on the neurons. On ciliary neurons, the α7-nAChRs are concentrated on somatic spines arranged in clumps and embedded in lipid rafts (Shoop et al., 1999, Shoop et al., 2002, Bruses et al., 2001). The α3*-nAChRs are concentrated in postsynaptic densities (Jacob et al., 1984, Wilson Horch and Sargent, 1995, Williams et al., 1998). Both can be activated by transmitter released from the large preganglionic calyx engulfing individual ciliary neurons (Zhang et al., 1996, Ullian et al., 1997, Coggan et al., 2005).

While both types of nicotinic receptors can participate in synaptic signaling in the ganglion (Chang and Berg, 1999), the α7-nAChRs due to their location and high relative calcium permeability (Bertrand et al., 1993, Seguela et al., 1993) also mediate local changes in calcium levels (Shoop et al., 2001). This calcium influx can have a number of consequences, including control of gene expression (Chang and Berg, 2001). Scaffold proteins, notably members of the PSD-95 family and APC, interact with either or both types of receptors (Conroy et al., 2003, Temburni et al., 2004, Farias et al., 2007), offering mechanisms for tethering signal-transducing components. And recently, the synaptogenic cell adhesion molecules neuroligin and L1 have both been shown to be expressed by CG neurons, to co-localize with nicotinic receptors on the neurons, and to influence synapse formation/stabilization (Triana-Baltzer et al., 2006, Conroy et al., 2007, Ross and Conroy, in press). These components suggest a molecular organization at nicotinic synapses that has unexpected features in common with glutamatergic synapses.

We show here that CG neurons express EphB2Rs, which co-localize with α7-nAChRs on the cell surface. Though EphB2Rs do not appear to interact directly with the α7-nAChRs, EphBR activation physically constrains α7-nAChRs on the neurons and enhances their downstream signaling. These effects can be elicited by treating with an ephrinB1 construct which serves as an agonist of EphBRs, and they are specific for the α7-nAChR. Further, we identify a multi-component pathway of kinases necessary for the ephrinB/EphBR-mediated enhancement of α7-nAChR function.

Section snippets

EphB2Rs Co-Distribute with α7-nAChRs in Chick Ciliary Ganglia

EphB2Rs are expressed in the chick ciliary ganglion (CG) and reach their highest relative levels during the period of naturally-occurring neuronal cell death in the ganglion, i.e. embryonic day (E) 8-E14 (Landmesser and Pilar, 1974). This was seen by performing quantitative Western blot analysis on extracts prepared from CGs taken at various times and normalizing either for the amount of ganglionic protein or number of neurons present (Fig. 1). The EphB2R component was detected with an

Discussion

We show here for the first time that EphBRs participate in nicotinic signaling pathways, co-distributing with α7-nAChRs and enhancing their function. The ephrinB1/EphBR interaction with α7-nAChRs does not markedly affect receptor currents, and the receptors do not appear to interact directly. Nonetheless, ephrinB1/EphBR interactions partially protect α7-nAChRs against dispersal following spine retraction or lipid raft disruption. Functionally, ephrinB1/EphBR interactions enhance α7-nAChR

Western Blots

Western blot analyses were performed on freshly dissected and detergent-solubilized E8–E18 CGs as previously described (Conroy et al., 2003). The blots were probed with an anti-chick EphB2R Ab kindly provided by Dr. Elena Pasquale (Burnham Institute; La Jolla, CA). In addition to the affinity purification initially described (Holash and Pasquale, 1995), the EphB2R Abs used here were purified further by cross-absorption with a GST-fusion protein containing the relevant region of the EphB4R

Acknowledgments

Grant support was provided by the National Institutes of Health Grants NS12601 and NS35469 and by a Young Investigator grant from the Tobacco-Related Disease Research Program (15KT-0157 to Z.L.). We thank Elena Pasquale (Burnham Institute; La Jolla, CA) for generous gifts of anti-chick EphB2R Ab, and Xiao-Yun Wang for expert technical assistance.

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    1

    Current Address: J. David Gladstone Institutes, 1650 Owens Street, San Francisco, CA 94158.

    2

    Current Address: Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, 855 Monroe Ave., Memphis, TN 38163.

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