3.1 Cocaine and amphetamine regulated transcript
CART is a neuropeptide encoded by the CARTPT gene, which,
consists of three exons and two introns (Dominguez, 2006; Douglass &
Daoud, 1996). Alternative splicing of this transcript results in two
biologically active forms, CART42-89 and
CART49-89 in humans, corresponding to
CART55-102 and CART62-102 in rodents
(Kuhar et al., 2002). Despite decades of research on CART since its
initial isolation and sequencing in 1981 (Spiess et al., 1981), the
cognate receptor(s) for CART remains disputed, and CART remains an
orphaned ligand (Lau & Herzog, 2014; Ong & McNally, 2020). While CART
remains orphaned, it is suggested to signal via a Gi/ocoupled GPCR, linked to phosphorylation of the ERK pathway (Lakatos et
al., 2005; Somalwar et al., 2018).
CART is densely expressed in reward-related circuits critically involved
in AUD, including the hypothalamus, nucleus accumbens, central amygdala
and Edinger-Westphal nucleus (Jaworski et al., 2008; Koylu et al., 1998;
Millan & McNally, 2012; Walker et al., 2021), and has been heavily
implicated in a range of drug-related behaviours, including AUD (for
reviews see; Kuhar, 2016; Ong & McNally, 2020; Vicentic & Jones,
2007). Early studies identified an association between an intron 1
polymorphism of the CART gene and alcoholism in a Korean population
(Jung et al., 2004). While tools to probe the function of CART are
limited, several transgenic mouse lines have provided some insights.
Using two different transgenic CART KO mouse lines, CART KO mice show
reduced alcohol intake and preference in a two-bottle choice procedure
(Maddern et al., 2023; Salinas et al., 2014). Interestingly, sex
differences arise in restricted binge alcohol access, with male CART KO
mice showing increased but female CART KO mice decreased alcohol intake,
driven by bitter taste sensitivity in CART KO female mice, involving
CART signalling in the central nucleus of the amygdala (CeA; Maddern et
al., 2023). CART is also implicated in alcohol seeking behaviours.
Central infusions of CART peptide (CART55-102) reduced
context-induced reinstatement of alcohol seeking (King et al., 2010),
whilst neutralisation of CART signalling
(anti-CART55-102) in the CeA reduced stress-induced
reinstatement of alcohol seeking (Walker et al., 2021). This is likely
linked to the role of CeA CART in alcohol withdrawal-induced anxiety, as
CeA CART55-102 neutralisation reduced social anxiety
induced by alcohol withdrawal (Dandekar et al., 2008). Further,
CART-containing neurons in the arcuate nucleus are activated following
re-exposure to stimuli previously associated with alcohol availability
(Dayas et al., 2008). Together, these data highlight a role of CART in
critical aspects of AUD from taste, consumption, alcohol-induced
withdrawal and relapse. Despite the vast literature implicating CART in
a range of alcohol-related behaviours the exact neurobiological
mechanism(s) mediating CART function remain unclear and are severely
hindered by the lack of known cognate receptor(s) (Ong & McNally,
2020).
Recently, two orphaned GPCRs, GPR68 (Foster et al., 2019) and GPR160
(Yosten et al., 2020), have been proposed as putative CART receptors.
GPR68 is a ubiquitously expressed proton-sensitive receptor in brain
neurons (Wang et al., 2020), and is expressed in key regions where CART
terminals are located, including the striatum, amygdala and hippocampus.
Additionally, GPR68 holds many characteristics of a peptide-activated
GPCR (Foster et al., 2019). Indeed, CART(42-89)9-28, a
shorter variant of the CART protein, led to GPR68-dependent mass
redistribution responses, suggested to reflect numerous intracellular
events, including protein trafficking and receptor internalisation, with
both sub- and low-micromolar potencies (Foster et al., 2019).
CART(42-89)9-28, along with two other peptides
(Osteocrin33-55 and Corticotropin), acted as positive
allosteric modulators of GPR68 (Hauser et al., 2020). However, this
research remains limited, and it is unclear whether CART peptides are
able to stimulate GPR68 in the brain (Funayama et al., 2023). Of note,
the primary signalling pathways for GPR68 appear to be
Gs and Gq (Mogi et al., 2005), whilst
CART is thought to be via Gi/o coupled signal
transduction (Lakatos et al., 2005; Somalwar et al., 2018), suggesting
this is unlikely to be a cognate receptor for CART.
Another oGPCR, GPR160 has recently been posited as a cognate receptor
for CART, driven by observations that either a CART antibody or GPR160
antibody were able to attenuate CART55-102 induced
nociceptive responses in mice (Yosten et al., 2020). Additionally,
CART55-102 stimulated cFos mRNA (a marker of neuronal
activation) expression in KATOIII cells with endogenous expression of
GPR160, and exogenous CART55-102 co-immunoprecipitated
with GPR160 antibody in KATOIII cell lysates (Yosten et al., 2020).
Furthermore, CART55-102 stimulated ERK phosphorylation
in PC12 cells, the only known cell line with specific binding of CART
(Lin et al., 2011), which was attenuated via a GPR160 mRNA-targeted
small interfering RNA (siGPR160; Yosten et al., 2020). Subsequent work
found that injection of GPR160 antibody, prior to CART peptide
CART55-102, into the 4th ventricle
prevented exogenous CART peptide-induced reductions in food and water
consumption in rats (Haddock et al., 2021). Although these studies
provided promising evidence of GPR160 being a putative receptor of CART,
they did not assess, or report, the specific binding and/or affinity of
CART peptide to GPR160 (Haddock et al., 2021; Yosten et al., 2020).
Importantly, a recent study found that the GPR160 antibody did not
displace binding of either CART55-102 or
CART62-102, nor did it compete with the specific binding
site of the CART peptide in PC12 cells (Freitas-Lima et al., 2023).
Additionally, no GPR160 mRNA or protein was found in PC12 cells,
suggesting that CART binding in PC12 cells occurs via a different
receptor present in this cell line (Freitas-Lima et al., 2023).
Furthermore, saturation and competition binding assays in a THP1 cell
line with high endogenous GPR160 expression revealed no specific
binding, or competition, with CART peptide radioligands in THP1 cells,
strongly suggesting no presence of a CART receptor (Freitas-Lima et al.,
2023). Thus, the identity of a cognate CART receptor remains elusive and
further work is needed. Without these developments the full potential of
targeting the CART system as a treatment for many neuropsychiatric
disorders, including AUD, remains stalled.