NEUROTROPHIN ACTIVATION OF CATECHOLAMINE STORAGE VESICLE PROTEIN GENE EXPRESSION: SIGNALING TO CHROMOGRANIN A BIOSYNTHESIS S. K. MAHATA,*²‡ M. MAHATA,²‡ H. WU,²‡ R. J. PARMER²‡ and D. T. O’CONNOR²‡ ²Department of Medicine and Center for Molecular Genetics, Un iversity of California, San Diego, California, U.S.A. ‡Department of Veterans Affairs Medical Center, San Diego, California, U.S.A. Abstract––Nerve growth factor differentiates precursor cells into sympathetic neurons. Does acquisition of a ‘‘neuronal’’ phenotype after nerve growth factor involve biosynthesis of chromogranin A, the major soluble protein in chromaffin granule cores? Nerve growth factor activated chromogranin A gene expression 7.6-fold in PC12 pheochromocytoma cells, and similarly activated PC12-transfected mouse, rat or human chromogranin A promoter/reporter constructs. Chromogranin A promoter 5-deletions narrowed the nerve growth factor response element to a region from 77 to 61 bp upstream of the cap site, a region containing the chromogranin A cyclic AMP response element (TGACGTAA). Three different site-directed mutations of the cyclic AMP response element each reduced the nerve growth factor effect by >90%. Transfer of the cyclic AMP response element to a heterologous (thymidine kinase) promoter activated that promoter 5-fold after nerve growth factor, while transfer of a cyclic AMP response element point-gap mutant (TGA-GTAA) to a heterologous promoter abolished the nerve growth factor effect. These findings indicate that the cyclic AMP response element in cis is, at least in part, both necessary and sufficient to activate the chromogranin A gene. Chemical blockade of the nerve growth factor receptor TrkA or the mitogen-activated protein kinase pathway component MEK substantially diminished nerve growth factor-induced expression of chromogranin A. By contrast, the response of chromogranin A to nerve growth factor was not impaired after blockade of phospholipase C- or phosphoinositide-3 kinase. Chemical blockade of TrkA, Ras, MEK or mitogen-activated protein kinase similarly inhibited nerve growth factor activation of chromogranin A. Expression of constitutively activated Ras, Raf or MEK mutants increased chromogranin A promoter activity. Expression of dominant negative (inhibitory) mutants of Sos, Ha-Ras, Raf1, mitogen-activated protein kinase, ribosomal protein S6 serine kinase II (CREB kinase) or CREB (KCREB) each inhibited the nerve growth factor-induced increase in chromogranin A promoter activity. Thus, each component of the mitogen-activated protein kinase pathway is crucially involved in relaying the nerve growth factor signal in trans to the chromogranin A gene, in the following proposed sequence: nerve growth factor  TrkA  Shc/Grb2/Sos  Ras  Raf  MEK  mitogen-activated protein kinase  ribosomal protein S6 serine kinase II  CREB  cyclic AMP response element.  1998 IBRO. Published by Elsevier Science Ltd. Key words: chromogranin A, PC12, nerve growth factor, transcription, catecholamine, chromaffin granule. Chromogranin A, 6 the index member of the chromogranin/secretogranin secretory protein family, 12,77 is widely distributed in endocrine and neuroendocrine cells 77 as well as in brain. 77 Its functions include binding calcium 72 and catecho- lamines 72 within the catecholamine storage vesicle core, and generation of peptides biologically active in the extracellular space, such as pan- creastatin, 64 -granin, 29 vasostatin, 1 parastatin, 15 *To whom correspondence should be addressed. Support: Department of Veterans Affairs, National Insti- tutes of Health, and American Heart Association. A bbreviations: CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; CRE, cyclic AMP response ele- ment; CREB, cyclic AMP response element (CRE) bind- ing protein; ERK, extracellular signal relay serine/ threonine kinase (mitogen activated protein kinase, MAP kinase, MAPK); Grb2, adapter protein that bridges Shc to Sos; MAP, mitogen-activated protein; MEK, dual- specificity (threonine/tyrosine) MAPK kinase/ERK kinase (mitogen activated protein kinase kinase, MAP kinase kinase, MAPKK); MSV, murine sarcoma virus; NGF, nerve growth factor; NGFR, NGF receptor; Raf1, serine/threonine kinase (mitogen activated protein kinase kinase kinase, MAP kinase kinase kinase, MAPKKK, MEK kinase, MEKK); Ras, guanine nucleotide-binding/ exchanging protooncogene product; RskII, ribosomal protein S6 serine kinase II (pp90 rsk , CREB kinase); RSV, Rous sarcoma virus; Shc, Src homology domain- containing protein; Sos, member of a guanine nucleotide- releasing protein family that enhances the rate of GDP- GTP exchange on Ras; TK, thymidine kinase; TrkA, receptor tyrosine kinase (nerve growth factor receptor, NGFR). Pergamon N euroscience Vol. 88, No. 2, pp. 405–424, 1999 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+ 0.00 PII: S0306-4522(98)00225-5 405