Doing Protein Kinase C: Membrane Estrogen Receptor Signaling in a Neural Circuit

Female sexual behavior has been an exquisitely valuable model for developing an understanding of mechanisms of steroid action on nerve cells (1,2). The report by Dewing et al. (3) in this issue continues this grand tradition by identifying a set of biochemical pathways that mediate intracellular signaling stemming from interactions of estradiol with membrane binding sites. The work is of note from two points of view. The first is the inclusion of these novel signaling pathways coupled to membrane estrogen receptors with the more conventional intracellular mechanisms of estrogen action on reproduction. The second is the incorporation of these findings into a functional neuronal circuit mediating reproduction. For those new to this field, the acceptance of multiple forms of the estrogen receptor and even the existence of membrane estrogen receptors seems commonplace. Lost in today’s calm sea are the stormy history of dogmatic fervor and the battles defending each fundamentalist view of how estrogen exerts its biological actions. Jensen’s reviews of this history (4,5) are very revealing, tracing the transitions from the early beliefs that estrogen had an enzymatic-like action to the discovery and defense of the existence of intracellular receptors. The acceptance of intracellular receptors for estrogen (and other steroids of course) led to other debates about activated and inactivated receptor states, including the relocation of receptors in these states to different subcellular compartments (e.g. Refs. 6 and 7). This acceptance of intracellular receptors grew largely from the development of methodological approaches to visualize the receptors themselves using labeling of specific receptor antibodies (4) or by autoradiographic means to see the incorporation of estrogen into cells (8,9). In contrast, the existence of membrane steroid receptors was not so fondly embraced. These receptors are much like the dark side of the moon in that despite the weight of decades of physiological evidence (10), the inability to see the receptors has contributed to continued skepticism (and mystery). The appeal of the scientific approach taken in the research leading to this report is that the authors have taken a set of molecular processes identified in individual hippocampal neurons (11) and applied them to solving a fundamental question within a hypothalamic circuit. The model from hippocampal neurons suggests that estradiol-17β binds to conventional estrogen receptors that have been inserted into the cell membrane. The estrogen receptors in turn couple (directly or indirectly) with metabotropic glutamate receptors (mGluR; in this case mGluR1a), triggering signaling cascades associated with the glutamate receptor. Dewing et al. (3) took a systematic approach to investigating the role of protein kinase C (PKC) mediating the effects of estradiol and glutamate. The signaling pathway studied came from a combination of previous research and from the authors’ microarray analysis of estradiol-induced proteins in the arcuate nucleus. The arcuate nucleus sends projections to the medial preoptic area, and this circuit is part of a clearly larger neural pathway known to regulate the expression of female sexual behavior (termed lordosis in rodents). The functional endpoints included lordosis itself as well as internalization of the μ-opioid receptor in medial preoptic neurons. The rationale for this latter measure comes from the authors’ previous work showing that opioid release in the medial preoptic area is a key synaptic event regulating the expression of lordosis (12). Thus, μ-opioid receptor internalization serves as a proxy for increased synaptic opioid release, which in turn is a proxy for neural activation necessary for lordosis (when lordosis itself was not measured). Indeed, the results of the study supported the proposed model. Administration of either estradiol or an mGluR1a agonist produced opioid receptor internalization. Blocking PKC antagonized the effect mediated by the stimulation of either receptor. Finally, PKC activation mimicked the effects of estradiol or mGluR1a on opioid receptor internalization or female sexual behavior. The linchpin in all of this is the demonstration that estrogen receptors co-immunoprecipitated with mGluR1 in membrane fractions from the arcuate nucleus. Two key developments in this field loom on the horizon. The first is of course the visualization of membrane estrogen receptors. Here, Jensen’s (5) lesson of alternative approaches derived from the identification of intracellular receptors will be applicable, because little progress has been made in this arena. The other development will be the molecular details of the nature of the membrane interactions between estrogen receptors and other protein partners. Like the basic research and clinical impact of such a simple discovery as a method to immunostain the estrogen receptor, a similar discovery for membrane estrogen receptors is eagerly awaited.