(Received for publication, May 12, 1995; and in revised form, July 5, 1995)
From the
Three distinct cDNAs encoding the preproadipokinetic hormones I,
II, and III (prepro-AKH I, II, and III), respectively, of Locusta
migratoria have been isolated and sequenced. The three L.
migratoria AKH precursors have an overall architecture similar to
that of other precursors of the AKH/red pigment-concentrating hormone
(RPCH) family identified so far. The AKH I and II precursors of L.
migratoria are highly homologous to the Schistocerca gregaria and Schistocerca nitans AKH precursors. Although the L. migratoria AKH III precursor appears to be the least
homologous to the Manduca sexta, Drosophila
melanogaster, and Carcinus maenas AKH/RPCH precursors, we
favor the opinion that the L. migratoria AKH III precursor is
evolutionary more related to the M. sexta, D.
melanogaster, and C. maenas AKH/RPCH precursors than to
the AKH I and II precursors of S. gregaria, S.
nitans, or L. migratoria. In situ hybridization
showed signals for the different AKH mRNAs to be co-localized in cell
bodies of the glandular lobes of the corpora cardiaca. Northern blot
analysis revealed the presence of single mRNA species encoding the AKH
I precursor (570 bases), AKH II precursor (
600 bases), and
AKH III precursor (
670 bases), respectively. Interestingly, flight
activity increased steady-state levels of the AKH I and II mRNAs
(
2.0 times each) and the AKH III mRNA (
4.2 times) in the
corpora cardiaca.
Three peptide hormones with hyperlipemic activity, the
adipokinetic hormones I, II and III (AKH ()I, II and III;
see Table 1)(1, 2, 3) , are synthesized
by the glandular neurosecretory cells of the corpora cardiaca (CC) of
the migratory locust, Locusta migratoria. These peptides are
members of a large family of structurally related but functionally
diverse peptides (the AKH/RPCH family)(4) . In the adult
locust, the AKHs I and II are released into the hemolymph during flight
and are involved in the mobilization of lipid and carbohydrate from the
fat body to serve as energy substrates for the flight
muscles(4, 5, 6, 7) . Data on the
release and functioning of AKH III are lacking so far. Isolation and
characterization of CC peptides revealed that two other locust species, Schistocerca gregaria and Schistocerca nitans, each
contain two AKHs that are mutually identical(1, 8) ,
whereas Manduca sexta and Drosophila melanogaster each contain only one AKH (9, 10) (see Table 1).
Molecular biological studies have resulted in the characterization of the structure of the AKH/RPCH precursors (a signal peptide, AKH/RPCH, a Gly-(Lys/Arg)-Arg sequence, and an AKH/RPCH-associated peptide (AAP/RAP), in this order) of S. gregaria, S. nitans, M. sexta, D. melanogaster, and Carcinus maenas(11, 12, 13, 14, 15, 16, 17) .
The biosynthesis of the AKHs in S. gregaria has been elucidated in detail by O'Shea and co-workers(13, 18, 19, 20, 21) . The signal peptide is co-translationally removed from prepro-AKH, generating pro-AKH. Next, proteolytic processing, which is preceded by dimerization of two pro-AKHs (I/I, I/II, or II/II) via their single COOH-terminal Cys residues, gives rise to two AKHs (I and/or II) and one homo- or heterodimeric peptide consisting of two AAPs (I/I, I/II, or II/II), a so-called AKH precursor-related peptide, as end products. The biosynthesis of AKH I and II of L. migratoria proceeds via the same pathway(3) .
For the migratory locust, we now present three cDNA sequences, each encoding a different AKH precursor. The present data show that the AKH I and II precursors of L. migratoria are highly homologous to their S. gregaria and S. nitans counterparts. The AKH III precursor of L. migratoria appears to be more homologous to the L. migratoria, S. gregaria, and S. nitans AKH I and II precursors than to the M. sexta, D. melanogaster, and C. maenas AKH/RPCH precursors. In addition, we show that flight activity differentially increases the level of each prepro-AKH mRNA.
The reaction mixture for 3` RACE of the pro-AKH I and II cDNAs (PCR I) contained 4 µM degenerate AKH-A primer, 0.25 µM adaptor I primer, and 1 µl of cDNA pool. Amplification products were separated by agarose gel electrophoresis, and bands of approximately 380 and 300 bp were excised and eluted in 20 µl of TE buffer each. Either 1 µl of 20-fold diluted 380-bp fragment or 1 µl of 20-fold diluted 300-bp fragment was subjected to a nested PCR (PCR II) in a reaction mixture containing 4 µM degenerate AKH-A primer and 0.25 µM adaptor II primer (see Table 2).
The reaction mixture for 3` RACE of the pro-AKH III cDNA (PCR III) contained 0.5 µM degenerate AKH-B primer, 0.5 µM adaptor III primer, and 2 µl of cDNA pool. 2 µl of 50-fold diluted PCR III was subjected to a nested PCR (PCR IV) in a reaction mixture containing 0.5 µM degenerate AKH-C primer and 0.5 µM adaptor I primer. 2 µl of 50-fold diluted PCR IV was subjected to a nested PCR (PCR V) in a reaction mixture containing 0.5 µM degenerate AKH-C primer and 0.5 µM adaptor II primer (see Table 2).
Subsequently, the three different pro-AKH 3` RACE products were used to screen a L. migratoria CC-specific cDNA library. The nucleotide sequences of the longest AKH I, II, and III cDNAs and their deduced amino acid sequences are shown in Fig. 1Fig. 2Fig. 3. The AKH I cDNA consists of 363 bp, including an open reading frame of 189 nucleotides encoding the L. migratoria AKH I precursor. The AKH II cDNA consists of 367 bp, including an open reading frame of 183 nucleotides encoding the L. migratoria AKH II precursor. The AKH III cDNA consists of 438 bp, including an open reading frame of 231 nucleotides encoding the L. migratoria AKH III precursor.
Figure 1: Nucleotide sequence of the cDNA encoding the AKH I precursor of L. migratoria and the deduced amino acid sequence of the precursor. Nucleotides are numbered 5` to 3`, beginning with the first residue in the coding region for the adipokinetic hormone I. Amino acid residues are numbered with the first residue (Gln) of the hormone as 1. The asterisk indicates the stop codon. The nucleotides corresponding to the polyadenylation signal (AATAAA) are underlined.
Figure 2: Nucleotide sequence of the cDNA encoding the AKH II precursor of L. migratoria and the deduced amino acid sequence of the precursor. For further details see the legend to Fig. 1.
Figure 3: Nucleotide sequence of the cDNA encoding the AKH III precursor of L. migratoria and the deduced amino acid sequence of the precursor. For further details see the legend to Fig. 1.
Figure 4: In situ hybridization of corpora cardiaca of L. migratoria. Alternate transverse sections through the CC containing cell bodies that show in situ hybridization signals for the pro-AKH I mRNA (I), for the pro-AKH II mRNA (II), and for the pro-AKH III mRNA (III).
In
order to examine the full-length AKH mRNAs as well as to study the
effect of flight activity on the AKH gene expression, RNA was extracted
from CC of locusts that had flown for 1 h as well as from CC of resting
locusts. RNA blot analysis (Fig. 5) revealed that the mRNA
encoding the AKH I precursor clearly is the most predominant AKH
transcript expressed in the CC. In addition, flight activity caused the
steady-state levels of the AKH I and AKH II transcripts and the AKH III
transcripts to increase approximately 2.0 and 4.2 times, respectively.
Northern blot analysis also showed that the cDNAs encoding the AKH I,
II, and III precursors represent transcripts of 570,
600, and
670 bases, respectively.
Figure 5:
Northern blot analysis of L.
migratoria AKH precursor mRNAs. A, hybridization of RNA
isolated from CC of locusts that had flown for 1 h (lanes indicated with F) as well as from CC of resting locusts (lanes indicated with R) with pro-AKH I cDNA probe (I), with pro-AKH II cDNA probe (II), and with
pro-AKH III cDNA probe (III) after washing with 1 SSC,
0.1% SDS at 65 °C for 2
15 min. Indicated are the
0.16-1.77-kb RNA size markers (Life Technologies, Inc.). B, after stripping off the AKH cDNA probes, the membranes were
hybridized with an 18 S RNA cDNA probe and washed with 1
SSC,
0.1% SDS at 65 °C for 2
15 min for standardization of the
amount of RNA loaded in each lane.
Further processing of the three different L. migratoria AAPs seems to be very unlikely, because at least unprocessed L. migratoria AAPs I and II have been isolated in the form of homo- or heterodimers, linked via their COOH-terminal Cys residues(29) . In addition, also from S. gregaria and S. nitans, unprocessed AAPs can be isolated in the form of homo- or heterodimers. In both Schistocerca species this dimerization also has to precede the prohormone processing at the Gly-Lys-Arg or Gly-Arg-Arg sequences(7) .
The presence of multiple bioactive peptides within single precursors is commonly observed(30) . A consequence of such prohormone structures is that multiple companion peptides may coordinately be synthesized and released. If individual peptides within prohormones control different though related physiological and/or behavioral processes, this mode of synthesis and release may coordinate the component elements of a complex physiological and/or behavioral repertoire(31) . This situation is even more complex for the peptides derived from the AKH I and II prohormones; dimerization of two pro-AKHs (I/I, I/II, or II/II) followed by proteolytic processing may give rise to different ``bouquets'' of AKHs (I and/or II) in combination with homo- or heterodimeric peptides consisting of two AAPs (I/I, I/II, or II/II). Data on the possible formation of intra- or intermolecular disulfide bridges of pro-AKH III are lacking so far.
Figure 6: Comparison of AKH/RPCH precursors. A, the single-letter codes are used to designate amino acids. The three domains of signal sequence, AKH/RPCH, and AAP/RAP are set apart. Amino acids that represent the site for enzymic precursor cleavage and carboxyl-terminal amidation are joined to the AKH/RPCH sequence. Gaps (indicated by hyphens) were introduced optionally to achieve maximum similarity as well as taking into account conservative amino acid substitutions. B, UPGMA tree for the AKH precursors of L. migratoria (indicated by Lom AKH I, Lom AKH II, and Lom AKH III), S. gregaria (Scg AKH I and Scg AKH II), S. nitans (Scn AKH I and Scn AKH II), M. sexta (Mas AKH), and D. melanogaster (Drm AKH), and the RPCH precursor of C. maenas (Cam RPCH).
The results of the Northern blot
analysis revealed that the prepro-AKH I, II, and III cDNAs very likely
are full-length, assuming an average poly(A) tail of 200 nucleotides (Fig. 5). Interestingly, the ratio of steady-state AKH I, AKH
II, and AKH III mRNA levels seems to be similar to the ratio of the AKH
I, AKH II, and AKH III peptides (14:2:1) present in the CC of resting
locusts(3, 33) . Because each AKH may have a different
though related function, we reasoned that flight activity might induce
a differential pattern of expression of the AKH genes in the CC.
Indeed, a remarkable increase in the level of the AKH III transcript
(4.2 times) was found in comparison with the increase of the
levels of AKH I and II transcripts (
2.0 times each) (Fig. 5). Thus, the experiments suggest a stimulus-dependent
differential pattern of expression of the AKH genes in one type of
neurosecretory cell. The remarkable difference in flight-induced AKH
III versus AKH I and II mRNA increase may shed new light on a
possible role for AKH III during flight activity. In addition, these
results are in accordance with the observed enhancement of the
production of secretory granules by the trans-Golgi network in
flight-stimulated adipokinetic cells of L. migratoria(34) .
From the above experiments it may be concluded that the three different forms of AKH mRNA and as a result the three different forms of AKH precursors are co-expressed in the same cells of the corpora cardiaca of L. migratoria.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X86799[GenBank], X86800[GenBank], and X86801[GenBank].