Molecular and Cellular Endocrinology 214 (2004) 155–165 Forskolin-resistant Y1 adrenal cell mutants are deficient in adenylyl cyclase type 4 Abdallah Al-Hakim a , Xianliang Rui b , Jennivine Tsao b , Paul R. Albert c , Bernard P. Schimmer a,b,∗ a Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1L6 b Banting and Best Department of Medical Research, University of Toronto, 112 College St., Toronto, Ont., Canada M5G 1L6 c Ottawa Health Research Institute (Neuroscience), University of Ottawa, Ottawa, Canada K1H 8M5 Received 3 September 2003; accepted 21 October 2003 Abstract Four mutant clones independently derived from the Y1 mouse adrenocortical tumor cell line have adenylyl cyclase (AC) activities that are resistant to forskolin, a direct activator of AC. In this study the AC isoform composition of the forskolin-resistant mutants was examined in order to explore the underlying basis for the resistance to forskolin. As determined by Western blot and RT–PCR analysis, the four forskolin-resistant mutants all were deficient in AC-4; the levels of other AC isoforms (AC-1, AC-3 and AC-5/6) were comparable to the levels in parent Y1 cells. Transfection of one of the mutant clones with an AC-4 expression vector increased forskolin-stimulated cAMP signaling, and restored forskolin-induced changes in cell morphology and growth. Taken together, these observations indicate that AC-4 deficiency is a hallmark of the forskolin-resistant phenotype of these mutants and suggest that AC-4 is an important target of forskolin action in the Y1 adrenal cell line. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Adenylyl cyclase type 4; Forskolin; Y1 mouse adrenocortical tumor cells 1. Introduction To date, nine membrane-bound adenylyl cyclase (AC) isoforms have been identified in mammalian cells. These isoforms have tissue-specific patterns of distribution and are distinguished by their responses to G protein subunits, calcium and protein kinases A and C—properties that are thought to contribute to the orchestrated actions of a diverse group of hormones, neurotransmitters and drugs on their respective target tissues (see D’Aquila et al., 1991; Defer et al., 2000; Onda et al., 2001, for review). Specific func- tions, however, have been definitively assigned to only a few of the mammalian isoforms. Based largely on the pheno- types of AC-disrupted mice, critical roles have been demon- strated for: AC-3 in olfaction (Wong et al., 2000); AC-5 in motor function (Iwamoto et al., 2003); AC-1 and AC-8 Abbreviations: AC, adenylyl cyclase; IPTG, isopropyl--d-thio- galactoside; mc2r, ACTH-receptor; MEM, minimal essential medium ∗ Corresponding author. Tel.: +1-416-978-6088; fax: +1-416-978-8528. E-mail address: bernard.schimmer@utoronto.ca (B.P. Schimmer). in aspects of synaptic plasticity and memory (Wong et al., 1999); AC-8 in modulation of anxiety (Schaefer et al., 2000) and in Ca 2+ -stimulated cAMP accumulation in the parotid (Watson et al., 2000). Overall, the nine AC isoforms are topologically similar. They have two transmembrane domains, each comprised of six helical clusters, and a catalytic core formed by the two major cytoplasmic domains of the enzyme (Sunahara et al., 1996; Tang and Hurley, 1998). Forskolin activates eight of the nine AC isoforms by binding to the catalytic core of each enzyme and stabilizing the enzyme in a cat- alytically favorable conformation (Tesmer et al., 1997); AC-9 fails to respond to forskolin but can be converted to a forskolin-responsive form by a single amino acid substitu- tion within the catalytic core (Yan et al., 1998). Because of its ability to activate most AC isoforms, forskolin has been widely used to examine the roles of cAMP in hormone action and other aspects of cell regulation. As a model for cAMP-mediated signaling in the adrenal cortex, we have been investigating forskolin action in the Y1 mouse adrenocortical tumor cell line and in deriva- tive mutants with specific defects in the cAMP-signaling 0303-7207/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2003.10.066