Insect Biochemistry and Molecular Biology 30 2000 579–589 www.elsevier.comlocateibmb
cDNA cloning and expression of a hormone-regulated heat shock protein hsc 70 from the prothoracic gland of Manduca sexta
1
Robert Rybczynski , Lawrence I. Gilbert
Department of Biology, Coker Hall CB 3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Received 20 September 1999; received in revised form 10 February 2000; accepted 17 February 2000
Abstract
The brain neuropeptide prothoracicotropic hormone PTTH stimulates a rapid increase in ecdysteroid hormone synthesis that is accompanied by general and specific increases in protein synthesis, including that of a 70 kDa cognate heat shock protein hsc 70.
To further understand the possible roles of hsc 70, hsc 70 cDNA clones were isolated from a tobacco hornworm Manduca sexta prothoracic gland cDNA library. All sequenced clones were highly homologous to the Drosophila hsc 70-4 isoform. Manduca hsc
70 mRNA levels during the last larval instar exhibited a peak at the onset of wandering and a peak that coincided with the major pre-metamorphic peak of ecdysteroid synthesis. Manipulations of the glands’ hormonal milieu showed that hsc 70 mRNA levels
respond to 20-hydroxyecdysone, dibutyryl cAMP, PTTH and the JH analogue hydroprene. The protein and mRNA data suggest that hsc 70 could be involved in a negative feedback loop regulating assembly of the ecdysone receptor complex.
2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Heat shock protein; Ecdysteroid; Transcription; Prothoracicotropic hormone; Juvenile hormone
1. Introduction
The finding that Drosophila chromosomal puffing occurred in response to thermal and chemical stress led
to the discovery of the gene families coding for the pro- teins termed heat shock proteins hsps see Petersen and
Mitchell, 1985. Stress-induced puffing resulted in the transcription and translation of the hsps and the
repression of normal, tissue-specific protein synthesis. Since the first studies in Drosophila, it has become clear
that the heat shock response is conserved among organ- isms as diverse as bacteria and mammals, and also that
homologues to the stress-induced hsps exist, which are constitutively-expressed
and are
abundant cellular
components [heat shock cognates hscs]. Hscs partici- pate in a wide variety of intra-cellular processes, chiefly
through their ability to bind other proteins and facilitate correct folding andor mediate import into cellular
Corresponding author. Tel.: +
1-919-966-5535; fax: +
1-919-962- 1344.
E-mail address: rybczybiomass.bio.unc.edu R. Rybczynski.
1
The sequence has been deposited in the GenBank data base under accession AF194819.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 3 1 - X
organelles see Fink, 1999. Understanding when and how hsps and hscs function has grown into an area of
major study, which, despite a significant effort, is still replete with unanswered and barely addressed questions.
Among the most abundant hscs and hsps are those of approximately 70,000 molecular weight hsp 70 family.
Although hscs in this family are constitutively expressed, their synthesis can be influenced by a variety of extra-
cellular signals.
The specific
stimulation of
the expression of an hspc 70 protein by peptide hormones
or neurotransmitters, rather than the induction of a full suite of stress-response proteins, has been described for
a number of tissues and cell types. For instance, in the adrenal gland, hspc 70 expression is up-regulated by
both ACTH and a dopaminergic agonist Blake et al. 1991, 1993 and in a cultured hepatoma cell line, insulin
stimulates hsp 70 expression Ting et al., 1989. In Man- duca sexta, the tobacco hornworm, the brain neuropep-
tide, prothoracicotropic hormone PTTH stimulates the prothoracic gland to synthesize ecdysteroids see Gilbert
et al., 1996 and this process, which requires translation, is accompanied by a specific increase in the translation
of several proteins including an hsc 70 protein Rybczynski and Gilbert, 1994, 1995a,b.
580 R. Rybczynski, L.I. Gilbert Insect Biochemistry and Molecular Biology 30 2000 579–589
The function of hsc 70 in PTTH-stimulated ecdystero- idogenesis has proven difficult to determine. Hsc 70 pro-
teins participate in a variety of cellular processes in addition to their roles in protein folding and import into
organelles see Mayer and Bukau, 1998; Fink, 1999. For instance, hsc 70s are also involved in clathrin
uncoating Chappell et al., 1986 and in vertebrate ster- oid hormone receptor assembly and function see Pratt
and Toft, 1997. Hsc 70s are also abundant proteins such that PTTH-stimulated changes in abundance must be
measured against a very high background. For these reasons, a molecular approach was chosen, with the hope
that determining the hsc 70 isoform distribution in the prothoracic gland and other tissues during development
and under in vitro PTTH stimulation might afford further insight into the relationship between ecdysteroid syn-
thesis and hsc 70 synthesis. The data indicate that the chief hsc 70 expressed in the prothoracic gland is a Man-
duca homologue of the hsc 70-4 gene product of Droso- phila Perkins et al., 1990. The translation of this hsc
70 undergoes short-term up-regulation by PTTH in the gland but longer exposure to PTTH or PTTH analogues
and to 20-hydroxyecdysone 20E result in down-regu- lation of both protein and mRNA levels, suggesting a
role in the negative feedback control of ecdysteroid receptor complex assembly in the prothoracic gland see
Gilbert et al., 1997.
2. Methods and materials
