InteractiveFly: GeneBrief

Cholecystokinin-like receptor at 17D1: Biological Overview | References


Gene name - Cholecystokinin-like receptor at 17D1

Synonyms - CCK-like receptor at 17D1

Cytological map position - 17D1-17D3

Function - G-protein coupled receptor

Keywords - growth of neuromuscular junction, CNS

Symbol - CCKLR-17D1

FlyBase ID: FBgn0259231

Genetic map position - chrX:18601727-18620646

Classification - 7TM_GPCR_Srx: Serpentine type 7TM GPCR chemoreceptor Srx

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. This study demonstrates that cholecystokinin-like receptor (CCKLR), and Drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR (CCKLR-17D1 but not CCKLR-17D3) or dsk produce severe NMJ undergrowth, whereas overexpression of CCKLR causes overgrowth. Presynaptic expression of CCKLR is necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduce synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. These results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. The Drosophila larval neuromuscular junction (NMJ) has become a powerful system for studying the molecular mechanisms underlying synaptic development and plasticity, and many of the key synaptic proteins are evolutionarily conserved (Chen, 2012).

Genetic and molecular analysis in Drosophila has uncovered numerous molecules and pathways that regulate NMJ growth, including proteins required for cell adhesion, endocytosis, cytoskeletal organization, and signal transduction via TGF-β, Wingless, JNK, cAMP, and other signaling molecules. For example, previous studies revealed that increased cAMP levels led to a down-regulation of the cell adhesion protein FasII at synapses, and the activation of the cAMP response element binding protein (CREB) transcription factor to achieve long-lasting changes in synaptic structure and function. Despite the identification and characterization of these various positive and negative regulators, understanding of the networks that govern synaptic growth is still incomplete, with many of the key components and mechanisms yet to be uncovered and analyzed (Chen, 2012).

To search for new regulators of synaptic growth, a forward genetic screen was conducted for mutants exhibiting altered NMJ morphology. In this screen, a new mutation was discovered that exhibits strikingly undergrown NMJs, which indicates disruption of a positive regulator of NMJ growth. The affected protein was identified as cholecystokinin (CCK)-like receptor (CCKLR), a putative neuropeptide receptor that belongs to the family of G-protein coupled receptors (GPCRs) sharing a uniform topology with seven transmembrane domains. When activated by their ligands, neuropeptide GPCRs affect levels of second messengers such as cAMP, diacylglycerol, inositol trisphosphate, and intracellular calcium. Through activation of their cognate receptors, secreted neuropeptides mediate communication among various sets of neurons as well as other cell types to regulate several physiological activities, including feeding and growth, molting, cuticle tanning, circadian rhythm, sleep, and learning and memory. In general, neuropeptides act by modulating neuronal activity through both short-term and long-term effects. Short-term effects include modifications of ion channel activity and alterations in release of or response to neurotransmitters. Long-term effects include changes in gene expression through activation of transcription factors and protein synthesis. In contrast with the well-known effects of neuropeptide signaling on neuronal activity and the strength of synaptic transmission, regulation of synaptic growth and development by neuropeptides has not previously been clearly established (Chen, 2012).

This study demonstrates that CCKLR is required presynaptically to promote NMJ growth. Moreover, mutations of drosulfakinin (dsk), which encode the predicted ligand of CCKLR, cause similar NMJ undergrowth and interact genetically with CCKLR mutations, indicating that DSK and CCKLR are components of a common signaling mechanism that regulates NMJ growth. In addition to the morphological phenotypes caused by mutations of CCKLR and dsk, mutant larvae also exhibit deficits in synaptic function. Through phenotypic analysis of double mutant combinations, it was shown that DSK/CCKLR signals through the cAMP-PKA-CREB pathway to regulate NMJ growth. The results suggest a novel role for neuropeptide signaling in regulation of synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural plasticity of synapses in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Neuropeptides, whose effects have been extensively studied at NMJs, are usually described as neuromodulators because they modify the strength of synaptic transmission. For example, proctolin can potentiate the action of glutamate at certain NMJs in insects. However, involvement of neuropeptides in regulating neural development has not been well characterized. Recently, a C-type natriuretic peptide acting through a cGMP signaling cascade was found to be required for sensory axon bifurcation in mice (Schmidt, 2009), which suggests that neuropeptides may have a broader role in development than previously appreciated. The current studies demonstrate that DSK and its receptor, CCKLR, are strong positive regulators of NMJ growth in Drosophila (Chen, 2012).

DSK belongs to the family of FMRFamide-related peptides (FaRPs), which is very broadly distributed across invertebrate and vertebrate phyla. Originally identified in clams, FaRPs affect heart rate, blood pressure, gut motility, feeding behavior, and reproduction in invertebrates. They have been shown to enhance synaptic efficacy at NMJs in locust and to modulate presynaptic Ca2+ channel activity in crustaceans. In Drosophila, various neuropeptides derived from the FMRFa gene can modulate the strength of muscle contraction when perfused onto standard larval nerve-muscle preparations. To these previously described functions of FaRPs, this study adds a new role as a positive regulator of NMJ development (Chen, 2012).

Transgenic rescue experiments, RNAi expression, and overexpression of WT CCKLR demonstrate that CCKLR functions presynaptically in motor neurons to promote NMJ growth. Downstream components of this pathway were identified on the basis of known biochemistry of GPCRs and phenotypic interactions in double mutant combinations. GPCRs typically function by activating second messenger pathways via G proteins. Because loss-of-function mutations in dgs (which encodes the Gsα subunit in Drosophila) cause NMJ undergrowth, it is hypothesized that CCKLR signals through Gsα. Consistent with this idea, it was found that presynaptic constitutively active dgs overexpression rescues the NMJ undergrowth phenotype of CCKLR mutants. Conversely, dominant dose-dependent interactions were observed between CCKLR-null mutations and mutations of rut, which encodes an AC; or PKA-C1, which encodes a cAMP-dependent protein kinase, resulting in significant reductions in NMJ growth. These data place CCKLR together with the other genes in a common cAMP-dependent signaling pathway that regulates NMJ growth (Chen, 2012).

It is known that the AC encoded by rut is activated by Gsα, and on the basis of the results, it is proposed that Gsα is downstream of CCKLR signaling. However, the NMJ undergrowth in rut1, which is a presumptive null mutant, is not as severe as that of a CCKLR-null mutant. This is likely due to the fact that the Drosophila genome contains up to seven different AC-encoding genes, all of which are stimulated by Gsα. Presumably, one or more additional AC-encoding genes share some functional overlap with rut in regulation of NMJ growth. This idea is in good agreement with the results of Wolfgang (2004), who found that the NMJ undergrowth phenotype of rut1 is weaker than that of dgs mutants, which they also interpreted as an indication that multiple ACs are activated by the Gsα encoded by dgs (Chen, 2012).

The primary effector of this pathway is CREB2, a transcriptional regulatory protein that is activated upon phosphorylation by PKA. Consistent with the idea that CCKLR ultimately acts via activation of CREB, loss-of-function mutations of dCreb2 or neuronal overexpression of a dominant-negative dCreb2 transgene cause NMJ undergrowth similar to that of CCKLR-null mutants. Additionally, loss of one copy of dCreb2 in a CCKLR heterozygous background also causes NMJ undergrowth, and overexpression of WT dCreb2 fully rescues the NMJ undergrowth phenotype of CCKLR null, even leading to NMJ overgrowth. Thus, regulation of NMJ growth through the CCKLR signaling pathway is clearly mediated by dCreb2, whose activity is itself necessary and sufficient for regulating NMJ growth. This conclusion differs from another study that suggested that dCreb2 is required for NMJ function but not NMJ growth. One possible explanation for this discrepancy is that a weaker, inducible heat shock-driven transgene was used to express dCreb2 in the earlier work, whereas strong constitutive neuronal drivers were used this study. In any case, the current results demonstrate that in addition to its known role in NMJ function, CREB2 is also a strong positive regulator of NMJ growth and is likely to play a greater role in structural plasticity of synapses in learning and memory in Drosophila than previously suggested. This conclusion is consistent with a recent study (Koon, 2011) indicating that sprouting of type II larval NMJs in response to starvation is stimulated by a cAMP/CREB-dependent pathway via activation of an octopamine GPCR (Chen, 2012).

In addition to being undergrown, NMJs in CCKLR mutant larvae also exhibit a functional deficit. This is perhaps less straightforward than it might seem. Previous analyses of mutations affecting growth of the larval NMJ in Drosophila have shown that there is no simple correlation between the size and complexity of the NMJ and the amplitude of EJPs or amount of neurotransmitter release. These discrepancies arise because of various homeostatic compensatory mechanisms and because some of the affected signaling pathways alter synaptic growth and synaptic function in different ways via distinct downstream targets. For example, a mutation in highwire, which has the most extreme NMJ overgrowth phenotype described, is associated with a decrease in synaptic transmission. Wallenda mutations have been shown to fully suppress the overgrowth phenotype, but have no effect on the deficit in synaptic transmission (Chen, 2012).

In the case of CCKLR mutants, however, there appears to be a very good correspondence between the morphological phenotype and the electrophysiological phenotype: the reduction in the total number of active zones in CCKLR larvae as measured morphologically correlates very well with the reduction in quantal content that was observe. In addition, no difference in CCKLR mutants was detected in calcium sensitivity of transmitter release or in the size or frequency of spontaneous release events. Thus, the synaptic growth phenotype of CCKLR mutant NMJs is sufficient to account for the functional phenotype. However, the possibility cannot be ruled out that DSK/CCKLR signaling also exerts some modulatory effect on NMJ function that is distinct from its effect on NMJ development (Chen, 2012).

DSK is identified as the Drosophila orthologue of CCK, the ligand of CCKLR in vertebrates, on the basis of sequence analysis. The genetic analysis strongly supports the conclusion that DSK is the ligand of CCKLR at the larval NMJ. First, mutations of dsk and expression of dsk RNAi result in NMJ undergrowth phenotypes similar to that of CCKLR mutants. Second, loss of one copy of both dsk and CCKLR in double heterozygotes results in NMJ undergrowth. Third, heterozygosity for dsk does not further enhance the phenotype of a CCKLR-null mutant as expected if DSK regulates NMJ growth through its action on CCKLR. Fourth, overexpression of UAS-dsk does not rescue the undergrowth phenotype of a CCKLR-null mutant, but CCKLR overexpression can rescue NMJ undergrowth of a dsk hypomorphic mutant (Chen, 2012).

The discovery of an entirely novel role for neuropeptide signaling in NMJ growth raises several questions about how this signaling is regulated and the biological significance of this mechanism. Although answers to these questions will require much additional work, an immediate question is whether a paracrine or autocrine mechanism is involved. In the case of octopamine-mediated synaptic sprouting in response to starvation, both autocrine and paracrine signaling are involved in the sprouting of type II and type I NMJs, respectively (Koon, 2011). In an early immunohistochemical investigation, it was reported that DSK was detected in medial neurosecretory cells in the larval CNS that extended projections anteriorly into the brain and posteriorly to the ventral ganglion (Nichols, 1992). As it was not possible to obtain the original DSK antiserum and raising a new antiserum was not successful, the previous report has not been extended or confirmed. Instead, tissue-specific RNAi experiments were performed to examine the spatial requirement for DSK. Pan-neuronal dsk RNAi expression indicates that DSK expression in neurons is required to promote NMJ growth. In addition, C739-Gal4-driven dsk RNAi also causes NMJ undergrowth, whereas OK-Gal4-driven dsk RNAi in motor neurons does not. The expression pattern of C739-Gal4 overlaps with the DSK-positive cells previously identified by immunohistochemistry, which suggests that DSK produced by those neurosecretory cells is required for normal NMJ growth. Thus, from available data, it seems most likely that DSK is acting in paracrine fashion to regulate NMJ growth. However, further investigation will be necessary to determine the exact source of the DSK that promotes NMJ growth to fully understand how this neuropeptide regulates NMJ development (Chen, 2012).

Studies have demonstrated a role for CREB in long-term synaptic plasticity-structural changes in synaptic morphology that underlie the formation of long-term memories (Benito, 2010). This study shows that in addition to CREB's role in structural modification of synapses in response to experience after development is complete, it is also a key regulator of growth and morphology during development of the larval NMJ. Moreover, although CREB is the transcriptional effector for many GPCRs, the fact that NMJs in CCKLR mutants are as undergrown as those of CREB mutants suggests that DSK/CCKLR signaling is a major input to CREB during NMJ growth. Many of the genes encoding intermediate components of the pathway such as dnc, rut, and PKA also have effects on NMJ growth and development as well as on synaptic plasticity and learning and memory, further emphasizing an overlap between the mechanisms that regulate synaptic growth during development and those that regulate postdevelopmental structural synaptic plasticity. These results raise the possibility that DSK/CCKLR signaling also plays a role in long-term synaptic plasticity and learning as well as in synaptic development (Chen, 2012).


REFERENCES

Search PubMed for articles about Drosophila CCKLR

Benito, E. and Barco, A. (2010). CREB's control of intrinsic and synaptic plasticity: implications for CREB-dependent memory models. Trends Neurosci 33: 230-240. PubMed ID: 20223527

Chen, X. and Ganetzky, B. (2012). A neuropeptide signaling pathway regulates synaptic growth in Drosophila. J Cell Biol 196: 529-543. PubMed ID: 22331845

Koon, A. C., Ashley, J., Barria, R., DasGupta, S., Brain, R., Waddell, S., Alkema, M. J. and Budnik, V. (2011). Autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. Nat Neurosci 14: 190-199. PubMed ID: 21186359

Nichols, R. (1992). Isolation and expression of the Drosophila drosulfakinin neural peptide gene product, DSK-I. Mol Cell Neurosci 3: 342-347. PubMed ID: 19912877

Schmidt, H., Stonkute, A., Juttner, R., Koesling, D., Friebe, A. and Rathjen, F. G. (2009). C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci U S A 106: 16847-16852. PubMed ID: 19805384

Wolfgang, W. J., Clay, C., Parker, J., Delgado, R., Labarca, P., Kidokoro, Y. and Forte, M. (2004). Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila. Dev Biol 268: 295-311. PubMed ID: 15063169


Biological Overview

date revised: 7 May 2013

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