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Abstract
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.
Highlights
HT3) have been identified (5-HT3A and 5-HT3R subunit to be cloned (5-HT3B)) that assemble into homomeric (5-HT3A) and heteromeric (5HT3A؉5-HT3B) complexes
An analysis of chimeric 5-HT3A receptor(R)1⁄75-HT3BR constructs suggests the presence of elements downstream of the first transmembrane domain of 5-HT3B subunits that inhibit cell surface expression
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
Summary
5-HT, 5-hydroxytryptamine; GABAA, ␣-aminobutyric acid (type A); HA, hemagglutinin; ER, endoplasmic reticulum; BiP, immunoglobulin heavy chain binding protein; COPI, COPII, coatomer proteins I and II. A role for COPI and COPII recruitment of proteins into ER transport vesicles has been postulated To investigate the mechanisms involved in the ER retention of the 5-HT3BR, we have examined the assembly and surface expression of 5-HT3A1⁄75-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. The export of 5-HT3B from the ER appears to require the masking or exposure of other signals downstream from this ER retention signal
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