Cell surface expression of 5-HT3 receptors is controlled by an endoplasmic reticulum retention signal

Two subunits of the 5-hydroxytryptamine type 3 (5-HT3) have been identified (5-HT3A and 5-HT3B) that assemble into homomeric (5-HT3A) and heteromeric (5-HT3A+5-HT3B) complexes. Unassembled 5-HT3B subunits are efficiently retained within the cell. In this study, we address the mechanism controlling the release of 5-HT3B from the endoplasmic reticulum (ER). An analysis of chimeric 5-HT3A receptor(R)·5-HT3BR constructs suggests the presence of elements downstream of the first transmembrane domain of 5-HT3B subunits that inhibit cell surface expression. To investigate this possibility, truncated 5-HT3B subunits were constructed and assessed for their ability to access the cell surface in COS-7 and ts201 cells. Using this approach, we have identified the presence of an ER retention signal located within the first cytoplasmic loop between transmembrane domains I and II of 5-HT3B. Transplantation of this signal (CRAR) into the homologous region of 5-HT3A results in the ER retention of this subunit until rescued by co-assembly with wild-type 5-HT3A. The mutation of this ER retention signal in 5-HT3B (5-HT3BSGER) does not lead to cell surface expression, suggesting the presence of other signals or mechanisms to control the surface expression of 5-HT3BRs. The generation of truncated 5-HT3BSGER constructs confirmed that the CRAR signal does play an important role in the ER retention of 5-HT3B.

Previous Section
Next Section
The electrophysiological actions of serotonin (5-hydroxytrptamine; 5-HT)1 include the mediation of fast excitatory responses in neurons of the central and peripheral nervous systems. These responses are induced by the activation of ligand-gated ion channels (5-HT3Rs). Peripheral receptors are thought to modulate pain and intestinal and cardiovascular functions (1). In the central nervous system, 5-HT3Rs are important targets for the control of emesis induced post-operatively by chemotherapy and radiotherapy (2) and in the palliative care of patients with multiple sclerosis (3).

The first 5-HT3R subunit cloned (5-HT3A) is able to generate functional homomeric receptors in heterologous expression systems (4, 5). However, the conductance of these recombinant receptors is too small to be resolved directly (sub-picosiemens), and the receptors do not resemble many native neuronal 5-HT3Rs (9–17 pS) (6, 7). The second 5-HT3R subunit to be cloned (5-HT3B) (5, 8) is not able to generate functional homomeric receptors because of retention in the ER (9). However, upon the co-expression of 5-HT3A and 5-HT3B, 5-HT3B is rescued and expressed on the cell surface as a heteromeric complex with a channel conductance similar to that of most native receptors (9). Electrophysiological studies suggest that both homomeric and heteromeric 5-HT3Rs may co-exist within the same neuron (6, 8, 10, 11).

The 5-HT3Rs belong to the superfamily of transmitter-gated ion channels that includes the nicotinic acetylcholine, GABAA, and glycine receptors. The structural relationship of the 5-HT3Rs to the other members of this group suggests that their assembly may involve similar post-translational events (12). The assembly of GABAA receptors is directed by specific assembly signals within the amino-terminal extracellular domain (13–20). The export of receptors from the ER represents a critical checkpoint for surface expression, with quality control within the lumen of the ER performed by the chaperone proteins BiP, calnexin, and protein disulfide isomerase (9, 21, 22). Interaction with such proteins can cause the intracellular retention of immature proteins by virtue of ER retention signals within the chaperone molecules. In addition, cytoplasmically exposed ER retention signals within the cargo proteins themselves have been identified as elements that control protein export from the ER (23–27). A unifying mechanism for the regulation of ER export by these cytoplasmically localized signals is not known. However, a role for COPI and COPII recruitment of proteins into ER transport vesicles has been postulated (Refs. 28–31, but see Ref. 24).

To investigate the mechanisms involved in the ER retention of the 5-HT3BR, we have examined the assembly and surface expression of 5-HT3A·5-HT3B subunit chimeras and truncation mutants of 5-HT3B. We demonstrate that 5-HT3B possesses an ER retention signal capable of preventing homomeric cell surface expression. This “CRAR” signal requires masking by subunit interactions with the 5-HT3A subunit, regardless of whether it is present natively in the 5-HT3B subunit or recombinantly within the homologous position in 5-HT3A. In addition, the export of 5-HT3B from the ER appears to require the masking or exposure of other signals downstream from this ER retention signal.