Molecular and Physiological Characterization of a Receptor for d -Amino Acid-Containing Neuropeptides

Article abstract of DOI:10.1021/acschembio.8b00167

Neuropeptides in several animals undergo an unusual post-translational modification, the isomerization of an amino acid residue from the l-stereoisomer to the d-stereoisomer. The resulting d-amino acid-containing peptide (DAACP) often displays biological activity higher than that of its all-l-residue analogue, with the d-residue being critical for function in many cases. However, little is known about the full physiological roles played by DAACPs, and few studies have examined the interaction of DAACPs with their cognate receptors. Here, we characterized the signaling of several DAACPs derived from a single neuropeptide prohormone, the Aplysia californica achatin-like neuropeptide precursor (apALNP), at their putative receptor, the achatin-like neuropeptide receptor (apALNR). We first used quantitative polymerase chain reaction and in situ hybridization experiments to demonstrate receptor (apALNR) expression throughout the central nervous system; on the basis of the expression pattern, we identified novel physiological functions that may be mediated by apALNR. To gain insight into ligand signaling through apALNR, we created a library of native and non-native neuropeptide analogues derived from apALNP (the neuropeptide prohormone) and evaluated them for activity in cells co-transfected with apALNR and the promiscuous Gα subunit Gα-16. Several of these neuropeptide analogues were also evaluated for their ability to induce circuit activity in a well-defined neural network associated with feeding behavior in intact ganglia from Aplysia. Our results reveal the specificity of apALNR and provide strong evidence that this receptor mediates diverse physiological functions throughout the central nervous system. Finally, we show that some native apALNP-derived DAACPs exhibit enhanced stability toward endogenous proteases, suggesting that the d-residues in these DAACPs may increase the peptide lifetime, in addition to influencing receptor specificity, in the nervous system. Ultimately, these studies provide insight into signaling at one of the few known DAACP-specific receptors and advance our understanding of the roles that l- to d-residue isomerization play in neuropeptide signaling.

Full text of DOI:10.1021/acschembio.8b00167

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Molecular and Physiological Characterization of a
Receptor for D-Amino Acid-Containing Neuropeptides
James W. Checco, Guo Zhang, Wangding Yuan, Ke Yu, Siyuan Yin, Rachel H. Roberts-
Galbraith, Peter M. Yau, Elena V. Romanova, Jian Jing, and Jonathan V. Sweedler
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00167 • Publication Date (Web): 15 Mar 2018
Downloaded from http://pubs.acs.org on March 17, 2018
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Molecular and Physiological Characterization of a Receptor for
Neuropeptides
D-Amino Acid-Containing
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James W. Checco1, Guo Zhang2, Wangding Yuan2, Ke Yu2, Siyuan Yin2, Rachel H. Robertsꢀ
Galbraith3§, Peter M. Yau4, Elena V. Romanova1,5, Jian Jing2*, Jonathan V. Sweedler1,5*
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1Beckman Institute for Advanced Science and Technology, University of Illinois at Urbanaꢀ
Champaign, Urbana, U.S.A.
2State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of
Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and
Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing
University, Nanjing, China
3Department of Cell and Developmental Biology, Howard Hughes Medical Institute, University
of Illinois at UrbanaꢀChampaign, Urbana, U.S.A.
4Roy J. Carver Biotechnology Center, Protein Sciences Facility, University of Illinois at Urbanaꢀ
Champaign, Urbana, U.S.A.
5Department of Chemistry, University of Illinois at UrbanaꢀChampaign, Urbana, U.S.A.
§Present address: Department of Cellular Biology, University of Georgia, Athens, U.S.A.
* Corresponding authors: jingj01@live.com, jsweedle@illinois.edu
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Abstract
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Neuropeptides in several animals undergo an unusual postꢀtranslational modification: the
isomerization of an amino acid residue from the ꢀstereoisomer to the ꢀstereoisomer. The
resulting ꢀamino acidꢀcontaining peptide (DAACP) often displays higher biological activity
than its allꢀ ꢀresidue analogue, with the ꢀresidue being critical for function in many cases.
L
D
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D
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L
D
However, little is known about the full physiological roles played by DAACPs and few studies
have examined the interaction of DAACPs with their cognate receptors. Here, we characterized
the signaling of several DAACPs derived from a single neuropeptide prohormone, the Aplysia
californica achatinꢀlike neuropeptide precursor (apALNP), at their putative receptor, the achatinꢀ
like neuropeptide receptor (apALNR). We first used quantitative PCR and in situ hybridization
experiments to demonstrate receptor (apALNR) expression throughout the central nervous
system; based on the expression pattern, we identified novel physiological functions that may be
mediated by apALNR. To gain insight into ligand signaling through apALNR, we created a
library of native and nonꢀnative neuropeptide analogues derived from the apALNP (the
neuropeptide prohormone) and evaluated them for activity in cells coꢀtransfected with apALNR
and the promiscuous Gα subunit Gαꢀ16. Several of these neuropeptide analogues were also
evaluated for their ability to induce circuit activity in a wellꢀdefined neural network associated
with feeding behavior in intact ganglia from Aplysia. Our results reveal the specificity of
apALNR and provide strong evidence that this receptor mediates diverse physiological functions
throughout the CNS. Finally, we show that some native apALNPꢀderived DAACPs exhibit
enhanced stability toward endogenous proteases, suggesting that the
Dꢀresidues in these
DAACPs may increase peptide lifetime, in addition to influencing receptor specificity in the
nervous system. Ultimately, these studies provide insight into signaling at one of the few known
DAACPꢀspecific receptors and advance our understanding of the roles that
isomerization play in neuropeptide signaling.
Lꢀ to Dꢀresidue
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INTRODUCTION
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Polypeptides, including neuropeptides, often undergo postꢀtranslational modifications
(PTMs) that significantly influence their biological activity.1ꢀ3 One poorly understood PTM is the
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enzymeꢀcatalyzed isomerization of one amino acid residue from the
Lꢀstereoisomer to the Dꢀ
stereoisomer (Figure 1a) to form a
D
ꢀamino acidꢀcontaining peptide (DAACP).4ꢀ10 DAACPs
have been identified from diverse animals in multiple phyla, where they act as neuropeptides,6ꢀ9
hormones,10 and toxins4,11 and, in many cases, are significantly more biologically active than
their allꢀ
L
ꢀresidue analogues.7,8,12 DAACPs are difficult to identify by commonly used mass
spectrometry (MS)ꢀbased peptide characterization techniques because DAACPs and their allꢀ
L
ꢀ
residue epimers have identical masses. Methods have been recently developed to address this
challenge and enhance the identification of DAACPs.8,9,13ꢀ16 Nonetheless, not only are the full
functions of identified DAACPs still unknown, many DAACPs may remain unidentified. In
addition, although
Dꢀresidues have been shown to increase the protease resistance of synthetic
peptides12,17 and several DAACP toxins and toxin fragments,4,5 relatively few studies have
directly examined the influence of
CNS proteases.
Dꢀresidues on the stability of cellꢀcell signaling DAACPs to
The marine mollusk Aplysia californica is an excellent model for investigations of learning
and memory, neural circuits, neurochemistry, and neural signaling because it possesses a
relatively small number of neurons, many of which are easily identifiable and have wellꢀdefined
functions.8,9,18ꢀ23 Importantly, four DAACPs have been reported in Aplysia: GdFFDꢀOH,6,8
GdYFDꢀOH,9 SdYADSKDEESNAALSDFAꢀOH,9 and NdWFꢀNH2.7 (In this report, lowercase
“d” indicates the following residue is a
Dꢀresidue.) GdFFDꢀOH, GdYFDꢀOH, and
SdYADSKDEESNAALSDFAꢀOH each arise from postꢀtranslational processing of a single
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precursor, the Aplysia achatinꢀlike neuropeptide precursor (apALNP, Figure 1b, c). GdFFDꢀOH
and GdYFDꢀOH are active in the feeding and locomotor circuits, as assessed by
electrophysiology and behavioral experiments,8,9,24 whereas SdYADSKDEESNAALSDFAꢀOH
is inactive in these circuits. NdWFꢀNH2, which is not derived from apALNP, is cardioactive. For
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all three active DAACPs in Aplysia, the results of experiments with the allꢀ
L
ꢀresidue analogues
demonstrated that the
PTM for function.
Dꢀresidue is essential for bioactivity, highlighting the importance of this
Figure 1. (a) Generation of a DAACP by an
L/Dꢀisomerase. (b) Protein sequence of apALNP (Genbank:
AAW30457.1). Predicted peptides generated from prohormone processing25 of apALNP are underlined and listed in
panel (c). Note that SdYADSKDEESNAALSDFAEDꢀOH was isolated as SdYADSKDEESNAALSDFAꢀOH in a
prior report9 (see note in Supporting Information). For panels (b) and (c), peptides in bold were tested for apALNR
activation (see Table 1). Residues previously identified to be converted to Dꢀresidues are depicted in red.
Several peptide toxins and designed therapeutics bearing
Dꢀresidues interact with
mammalian neuropeptide receptors,11,26 but the ways in which endogenous DAACPs enact their
functions are less well understood, primarily because few receptors for cellꢀcell signaling
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DAACPs are known. As part of a recent study of G proteinꢀcoupled receptor (GPCR)
deorphanization in Platynereis, Bauknecht and Jékely27 identified related GPCRs from several
phyla, here called achatinꢀlike neuropeptide receptors (ALNRs), that were activated by DAACPs
derived from ALNP homologues. The authors showed that the Aplysia ALNR (apALNR, Figures
S1ꢀS2) was activated by GdFFDꢀOH, but not by GFFDꢀOH, suggesting that this receptor may be
the endogenous receptor for GdFFDꢀOH. To our knowledge, ALNRs are among the first GPCRs
identified that appear to recognize DAACPs as their native ligands. However, outside of
identifying this ligandꢀreceptor pair, little is known about the range of functions mediated
through ALNRs in the CNS.
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A critical examination of an ALNR, including its expression and ligand specificity, would
clarify the roles played by ALNPꢀderived DAACPs in the CNS. Here, we study the molecular
and physiological actions of Aplysia apALNPꢀderived peptides through apALNR. Overall, our
results represent one of the first detailed studies of a GPCR that appears to signal primarily
through a DAACP, and provide evidence that the functions of apALNPꢀderived DAACPs extend
beyond feeding and locomotor circuits to control other behaviors throughout the CNS.
Ultimately, our results expand the roles that DAACPs play in the Aplysia CNS, and help to
clarify the role of
Lꢀ to
D
ꢀresidue isomerization in both the function and biological stability of
these neuropeptides.
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RESULTS AND DISCUSSION
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Expression of apALNR Across the CNS. Understanding the tissue localization of receptor
expression (e.g., which specific cells transcribe mRNA for the receptor) is a prerequisite for
understanding the physiological functions enacted through the receptor.28,29 In Aplysia, regions
of the CNS (and even many specific neurons) are mapped to wellꢀknown physiological and
behavioral functions. Because GdFFDꢀOH is active in the feeding and locomotor circuits,8,24
controlled primarily by neurons in the buccal and pedal ganglia, respectively, we predicted
apALNR would be expressed in these ganglia. Whether apALNR is expressed in any other tissues
is not known.
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To gain insight into the relative expression of apALNP and apALNR across different
tissues, we performed qPCR experiments to measure gene expression for each major ganglion in
the CNS (buccal, cerebral, pedal, pleural, and abdominal, Figure 2a and Figures S3–S5), as well
as other nonꢀCNS tissues (the buccal mass and gill). In contrast to the pedal gangliaꢀspecific
expression for apALNP (Figure 2b), which is consistent with previous in situ hybridization (ISH)
experiments,8 we observed that apALNR mRNA was present at relatively uniform levels across
each ganglia of the CNS (Figure 2c). As expected for a GPCR,30 the expression level of apALNR
is low compared to the neuropeptide prohormone apALNP, whose products are targeted for
release and are generally not recycled.
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Figure 2. (a) Cartoon representation of the Aplysia CNS, highlighting the relative positions of each major ganglia
and major connective nerves (cartoon not drawn to scale). (bꢀc) Relative expression of (b) apALNP (neuropeptide
prohormone) and (c) apALNR (receptor) in various tissues, as determined by qPCR. Buccal mass and gill were
included as nonꢀneural tissue. Note the differences in the vertical axes. Individual points represent each of three
biological sets of five animals. Bars represent mean ± standard deviation (SD) of these three biological sets.
To investigate the cellular localization of apALNR, we performed ISH using digoxigeninꢀ
labeled riboprobes that bind apALNR mRNA. ISH is a useful qualitative technique to identify
specific cells that express a given transcript, and complements the quantitative information
gained from qPCR measurements. Consistent with gene expression measurements by qPCR, our
ISH experiments for apALNR mRNA revealed cellꢀspecific staining in the buccal, cerebral,
pedal, and pleural ganglia (Figure 3 and Figure S6). In the buccal ganglia, staining was most
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obvious in several of the large motor neurons located at the ventral surface. This staining in the
buccal ganglia is consistent with the activity reported for GdFFDꢀOH and GdYFDꢀOH in the
feeding network.8,9 In the cerebral ganglia, staining was most pronounced in the metacerebral
cells (MCCs) and the region corresponding to the H and right G clusters. Weaker staining was
observed on the outer lateral edge in the A/B cluster, and in a small number of stained cells in the
E clusters. In the pedal ganglia, expression was broadly distributed, with the most intense
staining in septa IIIb and IIIc of the dorsal surface, consistent with the effects of GdFFDꢀOH on
locomotor programs and locomotor behavior previously observed.9,24 Staining was weaker in
neurons of the pleural ganglia than in the pedal ganglia, except for noticeable staining of LPl1, a
giant neuron in the left pleural ganglion. The pattern of staining in the abdominal ganglia was not
as consistent between preparations as it was for other ganglia examined (Figure S7), which could
reflect technical issues or biological variability in expression between animals.
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Figure 3. Representative images showing the localization of apALNR mRNA by ISH across different ganglia in the
Aplysia CNS. Antisense probes revealed apALNR expression (left) while sense probes were included as negative
controls (right). (a) Buccal ganglia (caudal surface). (b) Cerebral ganglia (dorsal surface). MCCs highlighted with
arrows. (c) Pedal and pleural ganglia (dorsalꢀlateral view). LPed: Left Pedal. RPed: Right Pedal. LPl: Left Pleural.
RPl: Right Pleural. LPl1 highlighted with arrow. Scale bars: 500 ꢁm.
Together, the qPCR and ISH experiments confirmed the predicted expression of apALNR
in the buccal and pedal ganglia, but also revealed expression of apALNR in the cerebral, pleural,
and abdominal ganglia, regions that were not previously anticipated to express receptors for
apALNPꢀderived DAACPs. The cellular expression of apALNR immediately suggests that
apALNPꢀderived DAACPs may play additional roles beyond the specific feeding and locomotor
circuits previously identified.8,24 For example, the MCCs of the cerebral ganglia enhance the
strength of buccal muscle contractions and modulate the output of the central pattern generator
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(CPG) for biting movements.31,32 Cellꢀspecific expression of apALNR in the MCCs suggests that
apALNPꢀderived DAACPs may modulate these specific feeding behaviors through these cells.
Similarly, apALNR expression in LPl1 suggests that apALNPꢀderived DAACPs may help
regulate defensive mucus release, a behavior mediated by this neuron.33
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GdFFD-OH Increases Excitability of LPl1. The presence of the apALNR transcript in
LPl1, an easily identifiable cell asymmetrically present in the left pleural ganglion, made this cell
a good candidate for testing the activity of apALNPꢀderived DAACPs for physiological
functions outside of the feeding and locomotor circuits previously tested.8,24 Neuropeptides,
through activation of cellꢀsurface receptors, can increase or decrease the excitability of a neuron.
To evaluate the effect of GdFFDꢀOH on LPl1, which expresses apALNR, we evaluated the
excitability of the LPl1 neuron under different conditions by electrophysiology. In these
experiments, 3 s constant current pulses were applied to LPl1 every 60 s and LPl1 excitability
was measured by the number of spikes evoked by the current pulses during the 3 s. Consistent
with its positive staining by apALNR ISH, we found that the excitability of LPl1 was increased
by a perfusion of GdFFDꢀOH, but not by GFFDꢀOH, in electrophysiology experiments on
isolated pleuralꢀpedal ganglia preparations (Figure 4 and Figure S8). Recordings on LPl1 were
performed in highꢀdivalent saline, which limits polysynaptic influences. Of course, receptors
other than apALNR may also be activated by GdFFDꢀOH, contributing to the observed
modulation. Nevertheless, this finding confirms that apALNPꢀderived peptides can modulate the
activity of neurons outside of the feeding and locomotor circuits, including a cell type known to
function in defensive mucus release.
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Figure 4. LPl1 excitability (tested with 3 s current pulses every 60 s) after perfusion of isolated pleural and pedal
ganglia of Aplysia with (a) GdFFDꢀOH (F(3,12) = 59.33, p < 0.001, n = 5) or (b) GFFDꢀOH (F(3,15) = 1, p > 0.05,
n = 6) (See Figure S8). “Control”: activity before peptide perfusion. “Wash”: activity after washout of peptide. Bars
represent mean ± standard error of the mean (SEM). Repeated measures ANOVA and Bonferroni post hoc test: *, p
< 0.05; ***, p < 0.001. Recordings were made in highꢀdivalent saline.
Activation of apALNR and the Feeding Network by apALNP-Derived Peptides. We
next sought to determine whether peptides from apALNP other than GdFFDꢀOH were ligands
for apALNR. Postꢀtranslational processing of apALNP is predicted to give rise to several
peptides: GFFDꢀOH, GYFDꢀOH, GDASꢀOH, SYADSKDEESNAALSDFAEDꢀOH, GFFꢀNH2,
and YYGSꢀOH, and Nꢀ and Cꢀterminal peptides (Figure 1b, c).25 Of these, we have identified
GdYFDꢀOH and SdYADSKDEESNAALSDFAꢀOH as endogenous DAACPs in the CNS of
Aplysia, in addition to GdFFDꢀOH.8,9
We synthesized neuropeptides predicted to arise from cleavage of the apALNP (Figure
1b, c), along with DAACP analogues, to test for direct activation of apALNR in CHOꢀK1 cells
expressing apALNR. For peptides that have not been detected as DAACPs, predicted DAACP
analogues were designed with the
Dꢀresidue at position 2, the location of the
D
ꢀresidue in all
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known molluscan DAACPs. Three predicted Nꢀ and Cꢀterminal peptides, which lack structural
similarity to known DAACPs from mollusks, were not evaluated. To determine apALNR
activation in response to potential agonists, we transfected CHOꢀK1 cells with plasmids for the
expression of apALNR and also Gαꢀ16, a promiscuous Gα subunit that associates with most
GPCRs and activates the phospholipase C signaling pathway.34 Activation of the canonical Gαq
signaling was then measured using a commercially available assay that measures the
accumulation of IP1 in the presence of LiCl upon activation of phospholipase C.35 Consistent
with a prior report,27 we found that GdFFDꢀOH activates apALNR whereas GFFDꢀOH does not,
using the IP1 accumulation assay (Table 1 and Figure S9). Interestingly, we found that GdYFDꢀ
OH was a potent agonist, with potency virtually identical to that of GdFFDꢀOH. The high
apALNR potency for GdYFDꢀOH is consistent with the fact that this peptide displays high
biological activity in physiological networks associated with feeding and locomotor behavior,
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similar to GdFFDꢀOH.9 In contrast, GYFDꢀOH, the allꢀ
Lꢀresidue analogue, was completely
devoid of apALNR activity, indicating that the ꢀTyr residue is critical for receptor activation for
D
GdYFDꢀOH. Both SdYADSKDEESNAALSDFAEDꢀOH and SYADSKDEESNAALSDFAEDꢀ
OH did not activate apALNR up to 50,000 nM (Table 1).
We also tested the predicted neuropeptide diastereomers GdFFꢀNH2/GFFꢀNH2, GdDASꢀ
OH/GDASꢀOH, and YdYGSꢀOH/YYGSꢀOH for activation of apALNR using the IP1
accumulation assay. We have detected several of these peptides in homogenized ganglia extracts
by liquid chromatography (LC)ꢀelectrospray ionizationꢀMS or with single cell MALDI MS,8,9
but the chirality of the endogenous peptides is currently unknown. We found that none of the
structural variants of GFFꢀNH2, GDASꢀOH, or YYGSꢀOH that we tested activated apALNR
(Table 1), though some variants appeared to show minor activity at the highest concentrations
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tested (500,000 nM). Thus, the presence of a
D
ꢀresidue at position 2 in a short peptide, even one
5
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as structurally similar to GdFFDꢀOH as GdFFꢀNH2, is not sufficient to confer apALNR activity.
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Table 1. Primary sequences of select peptides predicted from processing of apALNP, along with associated
apALNR activation EC50 values, as determined by IP1 accumulation assay in CHOꢀK1 cells transiently transfected
with both apALNR and Gαꢀ16 (see Figure S9 for all doseꢀresponse curves). For apALNRꢀactive compounds, EC50
are the mean from n ≥ 3 independent experiments. See Table S1 for error associated with these measurements. For
apALNRꢀinactive compounds, the EC50 is listed as being greater than the highest concentration tested, from n ≥ 2
independent experiments showing no or negligible activity. Activity in the Feeding Network is determined by
electrophysiology measurements on intact buccal and cerebral ganglia (Figure 6, Figures S10–S11); “Active”:
perfusion of peptide induced statistically higher circuit activity relative to “control” conditions with no peptide
perfusion. “Not active”: no increase in activity was detected. “NT”: not tested. Feeding Network activity for
GdFFDꢀOH/GFFDꢀOH and GdYFDꢀOH/GYFDꢀOH are from previous reports.8,9
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apALNR EC50 (nM)
(IP1 accumulation assay)
30
Feeding Network
(Electrophysiology)
Active
Peptide
GdFFDꢀOH
GFFDꢀOH
GdYFDꢀOH
>200,000
30
Not active
Active
GYFDꢀOH
>500,000
>50,000
>50,000
>500,000
>500,000
>500,000
>500,000
>500,000
>500,000
Not active
Not active
Not active
Not active
Not active
NT
SdYADSKDEESNAALSDFAEDꢀOH
SYADSKDEESNAALSDFAEDꢀOH
GdFFꢀNH2
GFFꢀNH2
YdYGSꢀOH
YYGSꢀOH
GdDASꢀOH
NT
NT
NT
GDASꢀOH
We previously found that GdFFDꢀOH and GdYFDꢀOH were both potent activators of the
CPG associated with feeding behavior in electrophysiology experiments, while their allꢀ
Lꢀ
residue analogues, GFFDꢀOH and GYFDꢀOH, were not (Table 1 and Figure S10).8,9,24 In the
present study, we evaluated the ability of SdYADSKDEESNAALSDFAEDꢀOH,
SYADSKDEESNAALSDFAEDꢀOH, GdFFꢀNH2, and GFFꢀNH2 to induce circuit activity in the
feeding network by electrophysiology. These electrophysiology experiments differ from the LPl1
experiments described above in that we measured the cyclic activity bursts of the I2 nerve of the
buccal ganglion and corresponding wellꢀdefined cycles of activity in several specific buccal
neurons that are known to lead to stereotyped feeding responses.36,37 When evaluated for activity
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in the feeding network, we found that all four peptides were inactive (Table 1, Figure S10, and
Table S1). Thus, for each peptide tested, in vitro activation of apALNR (or lack thereof)
determined by IP1 accumulation assay in cells transfected with apALNR completely matched
their physiological effects in the Aplysia feeding network.
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Together, our results support the identification of both GdFFDꢀOH and GdYFDꢀOH as
key apALNPꢀderived agonists of apALNR. The matching activity for apALNP peptides in
apALNR activation by IP1 accumulation assay and in the feeding network by electrophysiology
measurements supports the hypothesis that apALNR may mediate the effects of apALNPꢀderived
peptides in the feeding circuit. Furthermore, our results suggest that endogenous peptides, such
as GYFDꢀOH, SdYADSKDEESNAALSDFAEDꢀOH, and SYADSKDEESNAALSDFAEDꢀOH,
are inactive in the feeding network due to their inability to activate a key receptor, and not due to
other factors such as proteolytic degradation.
Activation of apALNR by GdFFD-OH Analogues. To gain more insight into how
apALNR recognizes its ligands and to determine if other peptides may be ligands for the
receptor, we designed a library of GdFFDꢀOH analogues bearing single residue substitutions or
terminal modifications, and evaluated the ability of each of these peptides to activate apALNR in
CHOꢀK1 cells transiently transfected with both apALNR and Gαꢀ16 using the IP1 accumulation
assay described above (Figure 5, Figure S9, and Table S2). The results, summarized in Figure 5,
reveal the specificity of the receptor and indicate that each residue of GdFFDꢀOH and both
terminal charges make important contributions to apALNR activity, as judged by apALNR
activation potency (EC50 values). For example, position 2 appears to require an extended
hydrophobic
Dꢀresidue, position 3 can tolerate smaller hydrophobic residues but not charged
residues, and position 4 requires a residue with a carbonyl group on its sidechain (see Supporting
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Information for more details). These results, which show that even relatively minor
modifications to GdFFDꢀOH lead to dramatic losses in potency in most cases, suggest that
GdFFDꢀOH and GdYFDꢀOH are likely the primary agonists for this receptor in vivo, although
unidentified or unrelated sequences may also be endogenous agonists.
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Figure 5. Primary sequences of GdFFDꢀOH analogues made through single residue substitutions (sub.) or terminal
modifications (mod.), along with associated EC50 values for apALNR activation (in parentheses) as determined by
IP1 accumulation assay in CHOꢀK1 cells transiently transfected with both apALNR and Gαꢀ16 (see Figure S9 for all
doseꢀresponse curves, and Table S2 for errors associated with these measurements). For nonꢀnatural analogues of
GdFFDꢀOH, the substituted residue/terminus is shown in red. For apALNRꢀactive compounds, EC50 is the mean
from n ≥ 3 independent experiments. For apALNRꢀinactive compounds, the EC50 is listed as being greater than the
highest concentration tested, from n ≥ 2 independent experiments showing no or negligible activity. EC50 values for
GFFDꢀOH, GYFDꢀOH, and GdYFDꢀOH, which are included in Table 1, are repeated here for comparison.
Physiological Activity of GdFFD-OH Analogues. To test the hypothesis that apALNR is a
mediator of the physiological effects of GdFFDꢀOH, we examined whether a subset of the
GdFFDꢀOH analogues described could directly induce feeding circuit activity in isolated buccal
and cerebral ganglia. For these studies, we chose GdFFDꢀOH analogues with high apALNR
potency (dAdFFDꢀOH), intermediate potency (GdLFDꢀOH, GdFADꢀOH, AcꢀGdFFDꢀOH, and
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GdFFDꢀNH2), or no activity (AdFFDꢀOH) in the IP1 accumulation assay. Consistent with its
high apALNR potency in the IP1 accumulation assay, we found that dAdFFDꢀOH activated the
Aplysia feeding network in a manner similar to GdFFDꢀOH and GdYFDꢀOH (Figure 6 and
Figure S11).8,9 GdLFDꢀOH, GdFADꢀOH, AcꢀGdFFDꢀOH, and GdFFDꢀNH2 were each weaker at
inducing circuit activation than dAdFFDꢀOH, consistent with their reduced apALNR potency
relative to dAdFFDꢀOH in the IP1 accumulation assay. Interestingly, we found that AdFFDꢀOH,
which showed no ability to activate apALNR in the IP1 accumulation assay experiments, was
modestly active in the feeding network. This contrasts with several other neuropeptides evaluated
(e.g., GdFFꢀNH2 and SdYADSKDEESNAALSDFAEDꢀOH, described above), which were
unable to activate apALNR by IP1 accumulation assay and showed no activity in the feeding
circuit. The inconsistency between apALNR activation in cellꢀbased assays with transiently
transfected receptor and electrophysiology activity in intact ganglia for AdFFDꢀOH might
indicate the inability of the apALNR IP1 accumulation assay to completely recapitulate the
complex biological interactions present in living neural networks. Indeed, peptide potencies can
differ significantly among different expression systems and intact tissues.38,39
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Figure 6. Feeding circuit activity induced by GdFFDꢀOH analogues (10ꢀ6 M or 10ꢀ5 M), as determined by
electrophysiological recordings on intact buccal and cerebral ganglia (see Figure S11). “Control”: activity before
peptide perfusion. “Wash”: activity after washout of peptide. Bars represent mean ± SEM. dAdFFD: F(3,18) =
12.51, p < 0.001, n = 7; GdLFDꢀOH: F(3,15) = 8.056, p < 0.01, n = 6; GdFADꢀOH: F(3,18) = 30.74, p < 0.001, n =
7; AcꢀGdFFDꢀOH: F(3,21) = 14.68, p < 0.001, n = 8; GdFFDꢀNH2: F(3,21) = 26.13, p < 0.001, n = 8; AdFFDꢀOH:
F(3,15) = 12.5, P < 0.001, n = 6. Repeated measures ANOVA and Bonferroni post hoc test: *, p < 0.05; **, p < 0.01;
***, p < 0.001.
Alternatively, activity for AdFFDꢀOH in the feeding network may indicate that there exist
alternative or modified isoforms of apALNR in vivo with altered selectivity, or that additional
unrelated receptors are activated by AdFFDꢀOH. Overall, the concordant activity for the
analogues tested, both for receptor activation by IP1 accumulation assay and in the feeding
network by electrophysiology (including apALNPꢀderived peptides, described above), is
consistent with apALNR as a mediator of the biological activity of this family of DAACPs,
although the unexpected activity of AdFFDꢀOH in the feeding network leaves open the
possibility that additional receptors for apALNPꢀderived DAACPs may also be present.
Stability of DAACPs in CNS Homogenates. Dꢀresidues are known to enhance the stability
of engineered peptides to proteases,5,12,17 but little is known about how
D
ꢀresidues influence the
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lifetime of naturally occurring DAACPs that act as cellꢀcell signaling peptides. To gain insight
into the relative stability of DAACPs from Aplysia to endogenous proteases, we incubated
exogenous GdFFDꢀOH, GdYFDꢀOH, GFFDꢀOH, GYFDꢀOH, and NdWFꢀNH2 in ganglia
homogenate extract and monitored the timeꢀcourse of peptide degradation by LCꢀMS with
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multiple reaction monitoring (MRM) (Figure 7). We found that the allꢀLꢀresidue peptides GFFDꢀ
OH and GYFDꢀOH were relatively rapidly degraded, with halfꢀlives of 8.4 and 8.7 min,
respectively. NdWFꢀNH2, a cardioactive DAACP, was more stable under these conditions (halfꢀ
life = 27 min). Previous studies have shown that the stability of NdWFꢀNH2 is similar to NWFꢀ
NH2 in ganglia membrane fractions,40 so we did not evaluate NWFꢀNH2 in this experiment.
Interestingly, both GdFFDꢀOH and GdYFDꢀOH were even more stable than NdWFꢀNH2, with
halfꢀlives of 530 and 1100 min, respectively, in the ganglia homogenate.
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Figure 7. Stability of 50 ꢁM neuropeptides in Aplysia cerebral and abdominal ganglia homogenate extracts in PBS,
pH 7.4, as determined by LCꢀMRM. Each point represents the mean ± SD of three biological sets of two animals
each. Panels (a) and (b) show the same data plotted on different time scales, while panel (c) shows calculated halfꢀ
life values for each peptide.
Unlike classical neurotransmitters, neuropeptides are released from both synaptic and
nonꢀsynaptic sites and can travel relatively long distances (on the order of micrometers) from
their site of release to activate distal receptors.41 This volume transmission mode of signaling
within the CNS, and even hormonalꢀlike roles traveling longer distances, are possible for
neuropeptides that display high lifetimes in the extracellular space.42,43 Our results are consistent
with the hypothesis that the
Dꢀresidue in apALNPꢀderived DAACPs increases the lifetime of
these peptides to allow signaling across relatively long distances. However, these studies were
performed in the environment of soluble ganglia homogenate extract, which likely lacks
membraneꢀbound enzymes that normally degrade these compounds and contains intracellular
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enzymes that would not naturally come in contact with these compounds in vivo. Further studies
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will be required to assess if GdFFDꢀOH and GdYFDꢀOH indeed possess longer lifetimes or
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travel greater distances in vivo relative to allꢀ
our results show that GdFFDꢀOH and GdYFDꢀOH have greater stability in an environment
containing endogenous CNS proteases than their allꢀ ꢀresidue analogues and another known
DAACP (NdWFꢀNH2) from the same animal. This suggests that in some cases the ꢀresidue can
Lꢀresidue peptides of similar length. Nevertheless,
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L
D
greatly extend the lifetime of cellꢀcell signaling DAACPs, in addition to being critical for
receptor activation.
Conclusions. Prior to our work, Bauknecht et al.27 showed
DꢀPheꢀspecific activation of apALNR
by GdFFDꢀOH, but little else about this ligandꢀreceptor family has been studied. Although
previous studies44,45 have explored how DAACP structure relates to specific physiological
functions (e.g., neuronal activity or muscle contraction), the receptors for most cellꢀcell signaling
DAACPs have not been identified, and thus, little is known about the interactions of DAACPs
with their receptors throughout the CNS. Here, we provide multiple compelling lines of evidence
demonstrating that apALNR does indeed function as a receptor for the apALNPꢀderived
DAACPs GdFFDꢀOH and GdYFDꢀOH, and mediates multiple physiological effects throughout
the CNS. First, qPCR and ISH analyses demonstrate, for the first time, that apALNR is expressed
in the Aplysia CNS. Interestingly, whereas apALNP expression is largely restricted to the pedal
ganglia, apALNR expression is distributed throughout the CNS. These results indicate that
apALNPꢀderived DAACPs may be produced in one central ganglion (i.e., pedal) to affect targets
throughout the CNS. Indeed, the apALNR expression data enabled us to identify a novel cellular
target of apALNPꢀderived DAACPs (LPl1), in addition to known targets in feeding and
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locomotor networks.8,24 The broad distribution of apALNR expression across the entire CNS
suggests that many other roles could be played by this interesting DAACPꢀreceptor family.
Receptor activation experiments with nonꢀnatural GdFFDꢀOH analogues (Figure 5) revealed that
each residue of this ligand makes important contributions to potency, and strongly suggest that
GdFFDꢀOH and GdYFDꢀOH are likely the primary ligands for apALNR. Finally, GdFFDꢀOH
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and GdYFDꢀOH were more stable than their allꢀ
extract, providing strong evidence that the lifetime of some cellꢀcell signaling peptides is
significantly increased in vivo due to the incorporation of the ꢀresidue, and may diffuse
Lꢀresidue analogues and NdWFꢀNH2 in ganglia
D
relatively long distances from their point of release throughout the CNS to activate distal
receptors.
The largely concordant activity of peptides in both receptor activation by IP1
accumulation assays and physiological experiments is consistent with the hypothesis that
apALNR mediates known physiological activities of GdFFDꢀOH and GdYFDꢀOH.8,9 However,
there may be other receptors that contribute to the physiological functions of GdFFDꢀOH and
GdYFDꢀOH. In fact, the physiological activity of AdFFDꢀOH in the feeding network, despite no
apALNR activity in the IP1 accumulation assay, suggests that there are additional receptors (or
isoforms of apALNR) with differing ligand specificities that are activated by GdFFDꢀOH or
GdYFDꢀOH. In the future, it will be of great interest to knockꢀdown apALNR expression to
determine the specific contributions of this receptor to single neuron excitability and/or network
activity. Regardless of whether apALNR is the sole receptor for GdFFDꢀOH and GdYFDꢀOH,
our results provide strong evidence that GdFFDꢀOH and GdYFDꢀOH are major ligands for
apALNR, and that apALNR is expressed across many regions of the CNS, including in regions
associated with feeding (buccal ganglia) and locomotion (pedal ganglia),8,9 and in specific cells
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known to control additional physiological functions, ultimately suggesting that GdFFDꢀOH and
GdYFDꢀOH play roles in a variety of physiological functions.
7
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Despite extensive efforts, there are still a large number of orphan GPCRs in vertebrates,
including mammals.46 The identification of ALNRs as receptors whose endogenous ligands
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require a
D
ꢀresidue for activation suggests that some orphan GPCRs may be selective for
ꢀ to ꢀresidue isomerization may
DAACPs, and deorphanization efforts that do not consider
L
D
miss ligandꢀreceptor identifications. Overall, our results represent one of the first inꢀdepth
explorations of a receptor that recognizes a DAACP as its native ligand, and provide evidence
that apALNPꢀderived DAACPs play a variety of functions throughout the CNS, either by
directly activating neurons or by acting as neuromodulators.41,47 Given the growing number of
DAACPs that continue to be identified among a wide variety of organisms,4ꢀ11 our findings may
be of significance for understanding
vertebrates.
Lꢀ to Dꢀresidue isomerization in other animals, including
METHODS
Detailed procedures can be found in the Supporting Information.
qPCR. qPCR experiments were performed using Power SYBR Green PCR Master Mix
(Applied Biosystems, 4368708), according to the manufacturer’s specifications. The relative
expression for each gene was calculated using the ꢂCt method.48 For each gene, ꢂCt was
calculated as the difference between the Ct values (mean of technical triplicates) for the gene of
interest and GAPDH. Relative expression values were calculated using 2ꢀꢂCt. See Supporting
Information for more details.
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In Situ Hybridization (ISH). The method for the ISH experiments was adapted from
Jezzini et al.49 using antiꢀsense or sense digoxigeninꢀlabeled RNA probes corresponding to a
~750 bp region of the apALNR mRNA (XM_005106549.2). Riboprobes were synthesized using
a SP6/T7 DIG RNA Labeling Kit (Roche), following the manufacturer’s instructions. See
Supporting Information for expanded details.
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Peptide Synthesis and Purification. The peptides GDASꢀOH and GdDASꢀOH were
purchased from CPC Scientific. All other peptides were synthesized by solidꢀphase peptide
synthesis based on Fmocꢀprotection of the main chain amine. Peptides with a Cꢀterminal acid
were synthesized on solid phase using Wang resin preꢀloaded with the Cꢀterminal residue
(Novabiochem or Anaspec). Peptides with a Cꢀterminal amide were synthesized on NovaPEG
Rink Amide resin (Novabiochem, 855047). Coupling reactions were carried out by treating the
resin with a solution of ≥4 molar equivalents Fmocꢀprotected amino acid with appropriate side
chain protecting groups, activated with benzotriazoleꢀ1ꢀylꢀoxyꢀtrisꢀpyrrolidinoꢀphosphonium
hexafluorophosphate (PyBOP) and N,Nꢀdiisopropylethylamine (DIEA) (1:1:2 molar ratio of
amino acid/PyBOP/DIEA) in a solution of 0.1 M NꢀHydroxybenzotriazole in Nꢀmethylꢀ2ꢀ
pyrrolidone. Coupling reactions were allowed to proceed at room temperature for greater than 40
min with stirring or gentle shaking, after which the resin was rinsed with 3–5 washes of
dimethylformamide (DMF). Deprotection of the Fmoc protecting group was carried out in a
solution of 20% piperidine in DMF for 20 min at room temperature with gentle stirring or
shaking, after which the resin was rinsed with 3–5 washes of DMF. Acetylation of the Nꢀ
terminus for AcꢀGdFFDꢀOH was achieved by treating the resin with a solution of 8:2:1
DMF/DIEA/acetic anhydride for 10 min. After the completion of the synthesis, peptides were
cleaved from the resin and sidechain protecting groups were removed using a solution of 95%
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trifluoroacetic acid, 2.5% H2O, and 2.5% triisopropylsilane for greater than 3 h. After cleavage,
most of the cleavage solution was removed by evaporation, and the peptides were dissolved in
water/acetonitrile (ACN) or dimethyl sulfoxide for HPLC purification. For
SdYADSKDEESNAALSDFAEDꢀOH and SYADSKDEESNAALSDFAEDꢀOH, peptides were
precipitated by the addition of cold methyl tertꢀbutyl ether. Crude peptide mixtures were purified
by reversedꢀphase HPLC and then dried under vacuum. Peptides were dissolved in a solution of
water/ACN and concentrations determined by UV absorbance and extinction coefficients
calculated from the primary sequence at 214 nm50 or 280 nm.51 Based on this calculated stock
concentration, peptides were aliquoted and dried under vacuum. Final peptide purity was
assessed by reversedꢀphase HPLC and identity confirmed by MALDIꢀTOF MS (see Supporting
Information).
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IP1 Accumulation Assays for apALNR Activation. CHOꢀK1 cells (ATCC, CLLꢀ61)
were transiently transfected with apALNR (region corresponding to XP_005106606.1, in
pcDNA3.1(+)) and Gαꢀ16 (in pcDNA3.1(+)) using Turbofect transfection reagent (ThermoFisher
Scientific, R0531). After exposure to potential agonist peptides for 1 h, activation of apALNR
was detected by monitoring IP1 accumulation using an IPOne Detection Kit (Cisbio,
62IPAPEB), following the manufacturer’s instructions with minor modification. While we found
that the recommended amount of IP1ꢀd2 and antiꢀIP1ꢀcryptate (1×) worked well, we also
obtained comparable EC50 values using half these amounts (0.5×), so many assays were
performed using 0.5× reagents. See Supporting Information for more details.
Electrophysiology. Intracellular and extracellular recordings of the physiological activity
from CNS preparations (either the cerebral and buccal ganglia, or the pleural and pedal ganglia)
were performed as described previously.8,52 Mean values were compared using repeated
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measures oneꢀway ANOVA, assuming sphericity, with the Bonferroni post test in GraphPad
Prism 7. See Supporting Information for notes on the statistical analyses and more details.
Peptide Stability Assay. Cerebral and abdominal ganglia were isolated from Aplysia
(65–85 g) and homogenized in phosphate buffered saline (PBS), pH 7.4. Each biological set
included cerebral and abdominal ganglia from two animals. Tissue was centrifuged (14,000 × g,
4 °C, 10 min) and the supernatant was removed and the protein content estimated using a BCA
Protein Assay Kit (ThermoFisher Scientific, 23235). Homogenate from each biological set was
diluted to 1000 ꢁg/mL protein. A stock solution containing 200 ꢁM of each peptide was prepared
in PBS. The peptide stock in PBS (125 ꢁL) was added to the ganglia homogenate (375 ꢁL) and
the resulting mixture incubated at 37 °C (final conditions = 50 ꢁM peptide, 750 ꢁg/mL
homogenate protein). At each time point, 10 ꢁL of the reaction mixture was removed and
quenched with 20 ꢁL of a solution containing 25 ꢁM GdFFꢀNH2 (as an internal control), 1%
formic acid (FA), in 50% ACN/water and stored at –20 °C until analysis. For analysis, each
quenched solution was thawed, diluted to 0.1% FA in 4% ACN/water, and desalted with C18
solidꢀphase exchange centrifuge spin columns (ThermoFisher Scientific, 89870). Desalted and
dried samples were dissolved in 0.1% FA in water and analyzed using a Bruker EVOQ Elite
tripleꢀquadrupole mass spectrometer coupled to a Bruker Advance UHPLC, MRM mode in
positive ion mode. Channels of parent and fragment ion pairs were identified using the MRM
Builder in the MS Workstation software for the EVOQ instrument, or were manually entered
based on predicted fragment ions. The parent ion (m/z, mass window: 0.7)/fragment ion (m/z,
mass window: 2) pair for each peptide were: GFFDꢀOH, GdFFDꢀOH = 485.2/120.2; GYFDꢀOH,
GdYFDꢀOH = 501.2/120.2; NdWFꢀNH2 = 465.2/448.6, GdFFꢀNH2 (synthesized with 13CꢀGly) =
370.2/120.1. For each time point, the peak area in the resulting LCꢀMRM chromatogram for the
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peptide of interest was first normalized to the peak area for GdFFꢀNH2, and the percent peptide
remaining subsequently calculated relative to the “1 min” time point for each biological set. Data
were plotted and halfꢀlife values calculated using a oneꢀphase exponential decay model in
GraphPad Prism 7.
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ASSOCIATED CONTENT
Supporting Information
Detailed materials and methods, notes on the apALNR characterization, statistical analyses, and
peptide selection/activity/inactivity, Tables S1 and S2, Figures S1–S11 (as noted in the text),
additional Figures S12–S14, peptide characterization data, and supporting references.
ACKNOWLEDGMENTS
We thank P. Bauknecht and G. Jékely for the generous gift of plasmids containing apALNR and
Gαꢀ16 for mammalian expression, and the UIUC Roy J. Carver Biotechnology Center for
providing resources, instrument time, and helpful advice. This work was supported by the
National Institutes of Health, Award No. P30 DA018310 from the National Institute on Drug
Abuse, and Award No. R01 NS031609 from the National Institute of Neurological Disorders and
Stroke (J.V.S.); the National Natural Science Foundation of China, Award Nos. 31671097,
31371104 (J.J.), and J1103512, and J1210026 (School of Life Sciences). J.W.C. was supported in
part by a Beckman Institute Postdoctoral Fellowship, funded by a Beckman Foundation gift to
the Beckman Institute for Advanced Science and Technology at the University of Illinois at
UrbanaꢀChampaign.
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