Neuronal calcium sensors emerge as novel adenylyl cyclase 8 regulators

Background

Adenylyl cyclases (ACs) are enzymes that catalyze the conversion of ATP to cAMP, an important second messenger required for a wide range of cellular functions. The ten members of the AC family are similar in their structural composition but differ in their functional characteristics and tissue distribution. Among them, AC1 and AC8 are activated by calcium (Ca²⁺) through the calcium binding protein calmodulin (CaM), conferring these members a unique function during neuronal development which is critically controlled by calcium signalling.

Beyond CaM, several calcium-sensing proteins contribute to Ca²⁺ dynamics and are involved in neuronal signalling. They are grouped under the neuronal calcium sensor (NCS) protein family, which is subdivided into five subfamilies including the visinin like proteins (VSNLs). This subfamily comprises visinin-like protein 1, 2 and 3 (VILIP-1, -2, and -3). VSNLs carry at their N-terminus a consensus motif that allows for N-myristoylation, a lipid modification that enables proteins to translocate from the cytosol to the membrane upon Ca²⁺ binding. This mechanism, known as the Ca²⁺-myristoyl switch, allows the interaction with target proteins present at the cellular membrane.

Previous studies reported a functional link between NCS and AC signalling; VILIP1 was shown to influence cAMP levels and to inhibit AC3 in Ca²⁺-dependent manner. These findings led the authors of this preprint to question whether the reported effects of NCS proteins on cAMP signalling are due to direct physical interactions with AC or indirect modulation of upstream cAMP signalling pathways.

 

What I like about the preprint

Having worked on HPCAL4 during my PhD, I’m keen to stay up to date with new research on the NCS protein family, which in my opinion remains underexplored despite their broad roles in neuronal physiology and association with some diseases. This preprint expands what is already known about the NCS family by identifying them as previously unrecognized regulators of AC8, thereby revealing a potential new role: Ca²⁺-dependent regulation of neuronal cAMP signalling.

It will be interesting to further explore how these interactions could contribute to signalling in a cellular context and whether they play a role in disease-related pathways. I really appreciated how the authors were transparent in acknowledging the limitations and challenges of the study. Rather than relying on a single approach, they combined complementary  experimental techniques ranging from biochemistry to integrative modelling to validate and strengthen their findings.

 

Key findings

HPCAL1 co-purifies with AC8 and binds in a calcium-dependent manner

In previous studies, AC8 has been shown to interact with calmodulin and heterotrimeric G proteins. To further extend the AC8 interactome, the authors expressed and purified AC8 from a monoclonal cell line and performed mass spectrometry. Dataset screening identified novel regulatory candidates among which HPCAL1 (VILIP-3) was highly abundant.
To understand if HPCAL1 interaction is regulated by Ca²⁺, the authors performed fluorescence detection size exclusion chromatography (FSEC) binding assays; in presence of EDTA, a Ca²⁺ chelator, HPCAL1 signal was abolished indicating that the interaction with AC8 is Ca²⁺-dependent.

HPCAL1 interacts with flexible regulatory regions of AC8

To determine which AC8 regions are required for interaction with HPCAL1, the authors expressed and purified the N-terminal, C-terminal and C1b domains individually and analyzed the interaction with HPCAL1 by nuclear resonance microscopy. The interaction was not detected with the isolated domains suggesting that full-length AC8 is required to establish the complex with HPCAL1.

These findings were further supported by FRET experiments in HEK293T cells co-transfected with HPCAL1 and AC8 deletion variants; FRET signal was reduced when AC8 was lacking either the N or C terminus. Furthermore, using MS-based approaches and integrative modelling, the authors identified a single region within AC8 C1b domain that exhibited a significant change upon addition of HPCAL1.

 AC8 establishes transient interactions with the NCS proteins

The authors hypothesized that other NCS family members might interact with AC8 since they share similar sequences. To study this, they expressed and purified additional NCS proteins (HPCAL4, VILIP1, HPCA, NCALD and NCS1). SEC-based in vitro binding assays revealed that these NCS proteins exhibit HPCAL1-like co-elution behavior with AC8, indicative of low-affinity interactions.

To further understand if these proteins could modulate AC8 activity, they performed an in vitro cAMP accumulation assay and found that only HPCAL4 elicited weak but significant activation of AC8 similar to that observed for HPCAL1.

 

Future directions and questions for the authors

• It can be challenging working with proteins that have a dynamic subcellular localization like the NCS. Given the transient interactions observed with AC8, have you considered performing co-immunoprecipitation or other complementary approaches to further validate these interactions in cells?
• It’s interesting that among the additional NCS proteins tested, only HPCAL4 was able to activate AC8 similarly to HPCAL1. What do you think distinguishes HPCAL4 from the other NCS proteins, and what implications might this have for the regulation of cAMP signalling ?
• Since the interaction between HPCAL1 and AC8 is calcium-dependent, do you anticipate that increasing intracellular Ca²⁺ levels would further enhance AC8 activation?

References

• Conti, A., Maas, J., Muglia, L., Muglia, L., Dave, B., Vogt, S., Tran, T., Rayhel, E., Muglia, L., & Muglia, L. (2007). Distinct regional and subcellular localization of adenylyl cyclases type 1 and 8 in mouse brain. Neuroscience, 146(2), 713–729. https://doi.org/10.1016/j.neuroscience.2007.01.045
• Ferguson, G. D., & Storm, D. R. (2004). Why Calcium-Stimulated adenylyl cyclases? Physiology, 19(5), 271–276. https://doi.org/10.1152/physiol.00010.2004

Tags: adenylyl cyclase, calcium, camp, proteomics

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