q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 191±200 191 Journal of Neurochemistry, 2001, 76, 191±200 Galanin receptor 1 gene expression is regulated by cyclic AMP through a CREB-dependent mechanism Venetia Zachariou, Dan Georgescu, Leena Kansal, Priscilla Merriam and Marina R. Picciotto Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, USA Abstract The galanin receptor-1 (GalR1) protein belongs to a family of G protein-coupled receptors for the neuropeptide galanin (GalR1, GalR2 and GalR3) distributed throughout the central and peripheral nervous system. Activation of galanin recep- tors by their ligands results in increased feeding, impaired learning, enhanced opiate analgesia and decreased opiate place preference. We have shown that opiate withdrawal, which is known to increase levels of cAMP in the locus coeruleus (LC), results in an increase in the number of galanin binding sites and the level of GalR1 mRNA in the LC. We have isolated a 3.6-kb fragment 5 0 of the inititiation codon of the mouse GalR1 gene and generated a series of deletion mutations of this fragment driving expression of luciferase for use in transient transfection assays in PC12 and Cath.a cell lines. Treatment with forskolin, but not dideoxyforskolin, up-regulates GalR1 transcription, likely through elevation of cAMP levels. The region between 2 1050 and 2 700 base pairs upstream of exon one is necessary both for basal activity of the GalR1 promoter and for forskolin-mediated increases in transcription. The forskolin effect can be blocked by simul- taneous mutation of a CRE-like site and a CRE/DRE-like site, but not mutation of either site alone. Gel shift and super-shift experiments demonstrate that the transcription factor CREB can bind to both sites and is likely to be responsible for the cAMP-mediated increase in GalR1 promoter activity. This study provides a molecular mechanism for the increased GalR1 expression in the LC seen following opiate withdrawal. Keywords: forskolin, luciferase, mouse, promoter, transfection. J. Neurochem. (2001) 76, 191±200. The neuropeptide galanin has effects on appetite, learning and nociception (McDonald and Crawley 1997). Galanin can also decrease the central reinforcing properties of morphine (Zachariou et al. 1999). Moreover, galanin is found at high levels in most norepinephrine-positive neurons of the locus coeruleus (LC), a brain area associated with drug dependence and withdrawal, and is able to decrease the ®ring rate of LC neurons in brain slices (Seutin et al. 1989; Sevcik et al. 1993; Pieribone et al. 1995). While galanin peptide mRNA levels do not change during withdrawal (Holmes et al. 1995), both galanin binding and galanin receptor 1 (GalR1) mRNA levels are increased in the LC following opiate withdrawal (Zachariou et al. 2000). Levels of both adenylate cyclase and cAMP increase in the LC following opiate dependence and withdrawal (Aghajanian 1978; Nestler 1992). Adaptive changes in receptors negatively linked to these effectors could be part of a regulatory mechanism to reduce the activity of these neurons. Three galanin receptors, GalR1, GalR2 and GalR3, have been identi®ed to date (Habert-Ortoli et al. 1994; Wang et al. 1997a, 1997b). GalR1 is expressed in many brain areas including the ventral hippocampus, olfactory tract, hypo- thalamus, nucleus accumbens and LC (Gustafson et al. 1996), whereas GalR2 is highly expressed in dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic and arcuate nuclei (Howard et al. 1997b; Wang et al. 1997a; Kolakowski et al. 1998), GalR3 is most highly expressed in the periphery whereas in the brain it is expressed in low abundance with the highest levels in the hypothalamus and pituitary (Smith et al. 1998). Activation of galanin receptors leads to inhibition of adenylate cyclase, and this effect appears to be responsible for the ability of galanin to Received July 17, 2000; revised manuscript received August 23, 2000; accepted August 29, 2000. Address correspondence and reprint requests to Marina R. Picciotto, Department of Psychiatry, Yale University School of Medicine, New Haven, CT 06508, USA. E-Mail: marina.picciotto@yale.edu Abbreviations used: BSA, bovine serum albumin; DDF, dideoxy- forskolin; GaIR1, galinin receptor 1; LC, locus coeruleus; NGF, ncerve growth factor; NRE, NGF-responsive element; TPA, 120-tetradecanoyl phorbol-13 acetate.