Non-full-length G protein-coupled receptors: An unexpected discovery may lead to design new molecular devices

It was believed that functionalities of G protein-coupled receptors (GPCRs) require full-length sequences which are negated by residue deletions. We demonstrate that significantly truncated nfCCR5QTY and nfCXCR4QTYstill bind their respective natural ligands. Receptor-ligand interactions were discovered from yeast-2-hybrid screening and confirmed by mating selection. Two nfCCR5QTY (SZ218a, SZ190b) and two nfCXCR4QTY(SZ158a, SZ146a) were produced in bacterial cell E. coli. Synthesized receptors exhibited a-helical structures, and bound respective ligands, albeit with reduced affinities. SZ190b and SZ158a were reconverted into non-QTY forms and expressed in HEK293T cells. Reconverted receptors localized on cell membranes and functioned as negative regulators for ligand-induced-signalling when co-expressed with full-length receptors. CCR5-SZ190b individually can carry out signalling, albeit at a reduced level with higher ligand concentration. Our findings provide insight into essential structural components for CCR5 and CXCR4 functionality, while raising the possibility that non-full-length receptors may be resulted from alternative splicing and that pseudogenes in genomes perhaps remain functional in living organisms.

Implications and future studies of truncated membrane receptors.

Our observation of non-full-length functional CCR5 and CXCR4 variants raises more questions than it provides answers.  For example: 1) Are there DNA sequences specifically coding for non-full-length receptors in all genomes? 2) Are they capable of performing regulatory functions in vivo at another level? 3) What are the smallest functional receptors that can exist in vivo? 4) Are they synthesized and subsequently cleared?

It is plausible that there are a few means of generating non-full-length receptors and proteins in general through: i) alternative RNA splicing; ii) SINE and LINE transposon insertions and deletions; iii) frame shift mutations resulting in premature translational termination; and iv) non-AUG translation initiation.  Many gene identification bioinformatics search for receptors and proteins with AUG as the translational initiation, and most experiments probe for RNA, rather than proteins. Therefore, it is plausible that such non-full-length proteins may have been overlooked. 

Our unexpected discovery of truncated membrane receptor variants in this study may thus alert us again to venture beyond current paradigms to discover, characterize, and design proteins.  Furthermore, our unexpected discovery may shed light to our knowledge blind spot, increase our understanding secrets of nature and open new opportunities to advance science in general.


Shuguang Zhang

Figure: The schematic illustration of the unexpected discovery for non-full-length G protein-coupled receptors and their function in either native (left) or soluble (right) forms. In native forms, the truncated receptors compete with full-length receptor for ligand binding and regulate calcium signaling. In soluble forms, the QTY variant receptors binds ligands in solution and act like decoy receptors as potential therapeutics. These non-full-length receptors are also useful for new bionic device design.