Final Report Summary - SIGT-OR-G (Signal transduction of Olfactory Receptors to their G protein)
Goal of project
Despite the incredible increase of structural information derived from X-ray diffraction data on G protein-coupled receptors (GPCRs) within the last years, the mechanistic details of GPCR mediated signal transduction remain elusive.
The present project concerned a specific class of GPCRs concentrating on the interaction of olfactory receptors (OR) and its G proteins during signal transduction. The particular goals were (i) to express a particular OR in a heterologous cellular system, (ii) to express the corresponding G protein Gαolf either in E.coli insect or in mammalian cell lines and eventually isolate the Gαolf, (iii) investigate the activation of the OR mediated signalling either in heterologous cells or in a reconstituted system by intervening the interaction between the OR and its Golf via competition with synthetic polypeptides of loop 2 of the OR.
Results
The human olfactory receptor OR5P3, as well as its homologue from mouse 204-6, was first expressed in HEK cell lines optimised for GPCR expression. pCRE-SEAP and pCRE GFP assays revealed the functional expression of the OR. In order to visualise the localization of the OR in living cells, the receptor was expressed as a fusion construct with a fluorescent protein. Despite of good cellular signalling activity, membrane localisation remained poor in all transfection experiments and the detergent extraction yielded only low amounts of the OR. Screens applying analytical size-exclusion chromatography with ultra-sensitive detection proved that the newly developed detergent NMG (Affymetrix) yielded the most monomeric OR, but still in low amounts. To validate the reliability of the applied expression system, other GPCRs were expressed for comparison and yielded substantially higher receptor concentrations in the cell plasma membranes, verifying that the chosen methods were in principle suitable for GPCRs. To circumvent day-to-day variations in receptor expression, cell lines stably expressing the receptor were established. The obtained clones showed excellent OR mediated signalling, but OR inserted in the cells’ plasma membranes and yields of the extracted receptors remained low. This is known from many reports of other ORs, the molecular reason however remains unresolved.
In order to investigate the binding of odorants to its OR directly we used a fluorescent analogue of an odorant molecule and thereby could detect the specific binding on the plasma membrane of a living cell directly.
To investigate the interaction between the OR and its Golf, we synthesized a peptide sequence, which is predicted to be directly involved in the OR-Gαolf complex. The peptide sequence was based on homology 3D models of the OR using existing high resolution GPCR structures as templates in combination with further sequence alignments. The peptides were synthesised by Fmoc strategy on solid support, purified by HPLC fluorescently labelled and again purified. The sequence of the synthesised peptides was proven by mass spectrometry.
For the production of Gαolf we constructed a G protein variant comprising an N-terminal affinity tag and expressed this Gαolf variant first in Escherichia coli using protocols developed for Gαs. As we did not succeed to express the protein in standard E. coli cell lines, we used E. coli strains optimised to express proteins at low temperatures. With this strategy we finally obtained soluble Gαolf. The production at low temperature were however much more time-consuming than initially foreseen. Therefore, we started production of Golf in mammalian and insect cell lines.
In parallel, we investigated whether ORs could be isolated together with its entire signalling machinery in cell-derived vesicles. Therefore we developed a proper procedure for purification and characterisation of such cell-derived vesicles. The results have been published in an article ‘Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy’ in Analytical Chemistry (Wyss R, Grasso L, Wolf C, Grosse W, Demurtas D, Vogel H (2014) Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy. Anal Chem 86 (15): 7229-7233.).
Although within the limited timeframe of this project, not all goals could be reached, important steps towards functional expression and purification of ORs and its G protein have been realised. The results are important milestones, which are presently used in follow-up projects of the host laboratory and more publications are expected to follow in this context.
Most importantly, the Marie Curie Fellow entered a new field of research and extended his technical experience and theoretical expertise substantially, as well as extending his professional network.
A side project yielded another publication on ‘Structure-based engineering of a minimal porin reveals loop-independent channel closure’ which was published in Biochemistry (Grosse W, Psakis G, Mertins B, Reiss P, Windisch D, Brademann F, Bürck J, Ulrich A, Koert U, Essen L-O (2014) Structure-based engineering of a minimal porin reveals loop-independent channel closure. Biochemistry 53 (29): 4826-4838.).
Despite the incredible increase of structural information derived from X-ray diffraction data on G protein-coupled receptors (GPCRs) within the last years, the mechanistic details of GPCR mediated signal transduction remain elusive.
The present project concerned a specific class of GPCRs concentrating on the interaction of olfactory receptors (OR) and its G proteins during signal transduction. The particular goals were (i) to express a particular OR in a heterologous cellular system, (ii) to express the corresponding G protein Gαolf either in E.coli insect or in mammalian cell lines and eventually isolate the Gαolf, (iii) investigate the activation of the OR mediated signalling either in heterologous cells or in a reconstituted system by intervening the interaction between the OR and its Golf via competition with synthetic polypeptides of loop 2 of the OR.
Results
The human olfactory receptor OR5P3, as well as its homologue from mouse 204-6, was first expressed in HEK cell lines optimised for GPCR expression. pCRE-SEAP and pCRE GFP assays revealed the functional expression of the OR. In order to visualise the localization of the OR in living cells, the receptor was expressed as a fusion construct with a fluorescent protein. Despite of good cellular signalling activity, membrane localisation remained poor in all transfection experiments and the detergent extraction yielded only low amounts of the OR. Screens applying analytical size-exclusion chromatography with ultra-sensitive detection proved that the newly developed detergent NMG (Affymetrix) yielded the most monomeric OR, but still in low amounts. To validate the reliability of the applied expression system, other GPCRs were expressed for comparison and yielded substantially higher receptor concentrations in the cell plasma membranes, verifying that the chosen methods were in principle suitable for GPCRs. To circumvent day-to-day variations in receptor expression, cell lines stably expressing the receptor were established. The obtained clones showed excellent OR mediated signalling, but OR inserted in the cells’ plasma membranes and yields of the extracted receptors remained low. This is known from many reports of other ORs, the molecular reason however remains unresolved.
In order to investigate the binding of odorants to its OR directly we used a fluorescent analogue of an odorant molecule and thereby could detect the specific binding on the plasma membrane of a living cell directly.
To investigate the interaction between the OR and its Golf, we synthesized a peptide sequence, which is predicted to be directly involved in the OR-Gαolf complex. The peptide sequence was based on homology 3D models of the OR using existing high resolution GPCR structures as templates in combination with further sequence alignments. The peptides were synthesised by Fmoc strategy on solid support, purified by HPLC fluorescently labelled and again purified. The sequence of the synthesised peptides was proven by mass spectrometry.
For the production of Gαolf we constructed a G protein variant comprising an N-terminal affinity tag and expressed this Gαolf variant first in Escherichia coli using protocols developed for Gαs. As we did not succeed to express the protein in standard E. coli cell lines, we used E. coli strains optimised to express proteins at low temperatures. With this strategy we finally obtained soluble Gαolf. The production at low temperature were however much more time-consuming than initially foreseen. Therefore, we started production of Golf in mammalian and insect cell lines.
In parallel, we investigated whether ORs could be isolated together with its entire signalling machinery in cell-derived vesicles. Therefore we developed a proper procedure for purification and characterisation of such cell-derived vesicles. The results have been published in an article ‘Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy’ in Analytical Chemistry (Wyss R, Grasso L, Wolf C, Grosse W, Demurtas D, Vogel H (2014) Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy. Anal Chem 86 (15): 7229-7233.).
Although within the limited timeframe of this project, not all goals could be reached, important steps towards functional expression and purification of ORs and its G protein have been realised. The results are important milestones, which are presently used in follow-up projects of the host laboratory and more publications are expected to follow in this context.
Most importantly, the Marie Curie Fellow entered a new field of research and extended his technical experience and theoretical expertise substantially, as well as extending his professional network.
A side project yielded another publication on ‘Structure-based engineering of a minimal porin reveals loop-independent channel closure’ which was published in Biochemistry (Grosse W, Psakis G, Mertins B, Reiss P, Windisch D, Brademann F, Bürck J, Ulrich A, Koert U, Essen L-O (2014) Structure-based engineering of a minimal porin reveals loop-independent channel closure. Biochemistry 53 (29): 4826-4838.).