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.