Molecular Interaction between Distal C-Terminal Domain of the CB₁ Cannabinoid Receptor and Cannabinoid Receptor Interacting Proteins (CRIP1a/CRIP1b)

Abstract

We have investigated the structure of the distal C-terminal domain of the of the CB1 cannabinoid receptor (CB1R) to study its interactions with CRIP1a and CRIP1b using computational techniques. The amino acid sequence from the distal C-terminal domain of CB1R (G417-L472) was found to be unique, as it does not share sequence similarity with other protein structures, so the structure was predicted using ab initio modeling. The computed model of the distal C-terminal region of CB1R has a helical region between positions 441 and 455. The CRIP1a and CRIP1b were modeled using Rho-GDI 2 as a template. The three-dimensional model of the distal C-terminal domain of the CB1R was docked with both CRIP1a as well as CRIP1b to study the crucial interactions between CB1R and CRIP1a/b. The last nine residues of CB1R (S464TDTSAEAL4722) are known to be a CRIP1a/b binding site. The majority of the key interactions were identified in this region, but notable interactions were also observed beyond theses nine residues. The multiple interactions between Thr418 (CB1R) and Asn61 (CRIP1a) as well as Asp430 (CB1R) and Lys76 (CRIP1a) indicate their importance in the CB1R–CRIP1a interaction. In the case of CRIP1b, multiple hydrogen bond interactions between Asn437 (CB1R) and Glu77 (CRIP1b) were observed. These interactions can be critical for CB1R’s interaction with CRIP1a/b, and targeting them for further experimental studies can advance information about CRIP1a/b functionality.

Deciphering the Molecular Association of Human CRIP1a with an Agonist-Bound Cannabinoid Receptor 1

“…Later, Singh et al 23 employed a similar combination of approaches and constructed an expanded 3D model depicting the interactions of the distal 56 residues of the C-terminal end of CB1 with a homology model of human CRIP1a. Although these models 23,24 provided insights into human CB1-CRIP1a interactions to some extent, it should be noted that the earlier studies modeled only parts of the Cterminal tail of CB1 while predicting their interactions with CRIP1a. However, it is expected that the presence of a lipidbound TM domain of CB1 in an active functional state could impact the conformational dynamics and relative positioning of its C-terminal tail, which were not taken into account in the previous models.…”

“…30 A C-terminal helix (Helix 8 in Figure 1a) of an activated CB1 couples with Gi 3 and G o and promotes the inhibition of cAMP, inhibition of Ca 2+ channels, inhibition of adenylate cyclase activity, activation of the mitogen-activated protein kinase (MAPK) pathway, and reduction of glutamate or neurotransmitter release into the presynaptic cleft. 23,24,28 However, binding of CRIP1a with CB1 changes its preference for Gi 3 and G o to Gi 1 and G 2 , 31 which in turn reduces the agonistmediated inhibition of Ca 2+ channels (especially the N-type channels) and increases the glutamate release. 12,28 Further, scientific evidence suggests that CRIP1a also competes with βarrestin and reverses the agonist-activated CB1 signaling.…”

“…To understand the binding mode of the CB1-CRIP1a complex, Ahmed et al 24 employed a combination of molecular modeling and protein−protein docking approaches and predicted a 3D model describing the interactions of the 9- residue long C-terminal tail of CB1 with a homology model of human CRIP1a. Later, Singh et al 23 employed a similar combination of approaches and constructed an expanded 3D model depicting the interactions of the distal 56 residues of the C-terminal end of CB1 with a homology model of human CRIP1a. Although these models 23,24 provided insights into human CB1-CRIP1a interactions to some extent, it should be noted that the earlier studies modeled only parts of the Cterminal tail of CB1 while predicting their interactions with CRIP1a.…”

“…Further, the human CRIP1a model in both the studies was constructed using the 3D structure of Rhospecific guanine nucleotide dissociation inhibitor 2 33 that only shares ∼16% sequence identify with that of human CRIP1a, which might have affected the accuracy of the predicted structures. 23,24 It is widely agreed that a minimum of 30% of template-target sequence identify is required to obtain a relatively good homology model. 34 Recently, the first mammalian crystal structure of rat CRIP1a 35 (PDB accession code: 6WSK) was reported in the PDB.…”

“…The C-terminal region of CB1 was found to be critical for G-protein coupling, desensitization, regulatory processes, and cellular trafficking of the receptor. − In particular, the C-terminal of CB1 interacts with different accessory proteins (Figure a) such as β-arrestins, adaptor protein complex-3 (AP3), G-protein-coupled receptor-associated sorting protein 1­(GASP1), factor associated with neutral sphingomyelinase activation (FAN), src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1), cannabinoid receptor interacting protein 1a (CRIP1a), and CRIP1b, which play significant roles in regulating the G-protein selectivity and signal transduction of CB1. ,, Therefore, exploring the recognition of these accessory proteins by CB1 has emerged as a novel avenue for understanding its signaling. In this work, we focus on building a comprehensive atomistic model describing the molecular association of the activated human CB1 with that of human CRIP1a. − …”