Er Alpha Delta 4, An Er Alpha Splice Variant Missing Exon4, Interacts With Caveolin-3 And Mglur2/3

The two isoforms of the nuclear estrogen receptor, ER alpha and ER beta are widely expressed in the central nervous system. Although they were first described as nuclear receptors, both isoforms have also been found at the cell membrane where they mediate cell signaling. Surface biotinylation studies using neuronal and glial primary cultures label an alternatively spliced form of ER alpha. The 52 kDa protein, ER alpha Delta 4, is missing exon 4 and is highly expressed in membrane fractions derived from cultured cells. In vivo, both full-length (66 kDa) ER alpha and ER alpha Delta 4 are present in membrane fractions. In response to estradiol, full-length ER alpha and ER alpha Delta 4 are initially trafficked to the membrane, and then internalized in parallel. Previous studies determined that only the full-length ER alpha associates with metabotropic glutamate receptor-1a (mGluR1a), initiating cellular signaling. The role of ER alpha Delta 4, remained to be elucidated. Here, we report ER alpha Delta 4 trafficking, association with mGluR2/3, and downstream signaling in female rat arcuate nucleus (ARH). Caveolin (CAV) proteins are needed for ER transport to the cell membrane, and using co-immunoprecipitation CAV-3 was shown to associate with ER alpha Delta 4. CAV-3 was necessary for ER alpha Delta 4 trafficking to the membrane: in the ARH, microinjection of CAV-3 siRNA reduced CAV-3 and ER alpha Delta 4a in membrane fractions by 50%, and 60%, respectively. Moreover, co-immunoprecipitation revealed that ER alpha Delta 4 associated with inhibitory mGluRs, mGluR2/3. Estrogen benzoate (EB) treatment (5 mu g; s.c.; every 4 days; three cycles) reduced levels of cAMP, an effect attenuated by antagonizing mGluR2/3. Following EB treatment, membrane levels of ER alpha Delta 4 and mGluR2/3 were reduced implying ligand-induced internalization. These results implicate ER alpha Delta 4 in an estradiol-induced inhibitory cell signaling in the ARH.