Membrane Assembly of the Cannabinoid Receptor 1: Impact of a Long N-Terminal Tail

Abstract

The human cannabinoid receptor 1 (CB1) belongs to the G protein-coupled receptor (GPCR) family. Among the members of GPCR family, it has an exceptionally long extracellular Nterminal domain (N-tail) of 116 amino acids but has no typical signal sequence. This poses questions of how the long N-tail affects the biosynthesis of the receptor and of how it is inserted into the endoplasmic reticulum (ER) membrane. Here we have examined the process of membrane assembly of CB1 in the ER membrane and the maturation of the receptor from the ER to the plasma membrane. We find that the long N-tail cannot be efficiently translocated across the ER membrane, causing the rapid degradation of CB1 by proteasomes; this leads to a low level of expression of the receptor at the plasma membrane. The addition of a signal peptide at the N terminus of CB1 or shortening of the long N-tail greatly enhances the stability and cell surface expression of the receptor without affecting receptor binding to a cannabinoid ligand, CP-55,940. We propose that the N-tail translocation is a crucial early step in biosynthesis of the receptor and may play a role in regulating the stability and surface expression of CB1.

Prolonged Recovery Rate of CB1 Receptor Adaptation after Cessation of Long-Term Cannabinoid Administration

“…The predicted molecular mass of unmodified CB 1 receptor protein is 53 kDa, but the major immunoreactive band was observed at an apparent mass of 45 kDa. This enhanced mobility of unglycosylated CB 1 receptor has been reported previously and is commonly observed for other membrane proteins as a result of their biophysical properties in SDS micelles (Andersson et al, 2003). There was another prominent band of 30 kDa in the hippocampus, whereas multiple higher mass forms were apparent in striatum/GP.…”

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…In the case of particular proteins (e.g., the GnRHR, the V2R, and rhodopsin), this approach has succeeded with a striking number of different mutants, supporting the view that pharmacoperones will become powerful ammunition in our therapeutic arsenal (Bernier et al, 2004b). On the other hand, it has also become clear that variable amounts of even some WT GPCRs are misrouted, presumably as a result of misfolding , 2001Andersson et al, 2003;Janovick et al, 2003b;Lu et al, 2003Lu et al, , 2004Cook et al, 2003;Pietilä et al, 2005), suggesting that this level of post-translational control may itself be amenable to pharmacological intervention and provide another level of potential therapeutic intervention (UlloaAguirre et al, 2006).…”

“…The further observation that pharmacoperones increase the PME of the WT human GnRHR itself, but not the rat counterpart, means that the human protein was only partially transferred to the PM and the remainder was apparently retained in the ER and eventually degraded, as has been found for other intracellularly retained GPCRs Andersson et al, 2003;Cook et al, 2003;Lu et al, 2003). Moreover, the observation that the human GnRHR is more susceptible to mutations than the rat or mouse ortholog supports the view that the human receptor is very precariously balanced between retention in the ER and routing to the PM and provides an underlying mechanism for a novel level of post-translational regulation of WT proteins (Conn et al, 2006a,b;Janovick et al, 2006).…”

“…Moreover, the observation that the human GnRHR is more susceptible to mutations than the rat or mouse ortholog supports the view that the human receptor is very precariously balanced between retention in the ER and routing to the PM and provides an underlying mechanism for a novel level of post-translational regulation of WT proteins (Conn et al, 2006a,b;Janovick et al, 2006). Retention of WT proteins is sometimes referred to as "inefficient" protein utilization and there is evidence that this mechanism is used by a variety of systems (Petäjá -Repo et al, 2001;Andersson et al, 2003;Cook et al, 2003;Lu et al, 2003). It is not seen in rat or mouse GnRHRs, which route very efficiently to the PM (Janovick et al, 2003b), suggesting that the expression of wild-type receptors that route heavily to the PM and are not retained by the ER QCS normally may be less sensitive to point mutations, as exemplified by the rat and mice GnRHRs.…”

Rapid CB1 cannabinoid receptor desensitization defines the time course of ERK1/2 MAP kinase signaling

“…Full-length rat CB 1 , with an amino-terminal pplssHA (prolactin signal sequence and hemagglutinin epitope) tag (Andersson et al, 2003) in pcDNA3.0 (Invitrogen, Carlsbad, CA), was used as a template with the following primers: forward 5′ ccgcacagcctcttgacaacgccatgggggacgcagactgcctgc-3′ and reverse 5′-gcaggcagtctgcgtcccccatggcgttgtcaagaggctgtgcgg-3′. A silent mutation was inserted (the 14 th nucleotide) to remove the XbaI site for screening purposes.…”