Researchers have developed a method to study how proteins are distributed on cell membranes. The method reveals that the spatial organisation of proteins is crucial for interpretating the signals sent into the cell.
G protein–coupled receptors (GPCRs) are as important to life as breathing.
This family of about 800 proteins is crucial for encoding cell-signalling responses from the environment into the interior of the cell.
GPCRs enable cells to respond to smell, taste, light and other extracellular stimuli, and 35% of all modern drugs therefore target GPCRs.
Until now, researchers’ knowledge about GPCRs had been incomplete, but a new study is plugging these gaps by developing a method to finally enable scientists to study how proteins are spatially organised at the cell membrane and to determine what factors determine the distribution of GPCRs there.
The discoveries open up new fields of research focusing on the effects of drugs or how cellular shape influences cell function.
“For more than 50 years, researchers have wrestled with questions that they have been unable to answer because they did not have the methods to study the organisation of GPCRs at the cell membrane, but we can do this now. Our results show that the shape of cells and the structure of the cell membrane are decisive in how GPCRs are spatially organised at the cell membrane and that the shape of cells strongly influences how cells interprets extracellular signals, and these are new,” explains a researcher involved in developing the method, Dimitrios Stamou, Professor, Center for Geometrically Engineered Cellular Systems, Department of Chemistry, University of Copenhagen.
The research has been published in Nature Chemical Biology.
Membrane proteins have been difficult for researchers
For decades, the challenge for researchers has been to understand how about 800 GPCRs can convert information from tens of thousands of extracellular stimuli into actions inside the cell.
GPCRs are seven-transmembrane-domain receptors, with part of the protein on the outside of the membrane facing the environment and part of the protein on the inside of the membrane facing the interior of the cell.
When a molecule binds to the receptor on the outside of the membrane, the protein changes shape, and this change results in a signal on the inside. A cell can thus adapt to certain situations by changing the protein expression in response to events in the extracellular environment.
Since the ratio is not 1:1:1 – one signalling molecule to one GPCR to one signal within the cell – researchers have long speculated that, for example, the spatial organisation of GPCRs in the cell membrane may play a role in expanding the repertoire of possible signalling pathways.
However, until now this theory could not be investigated.
“Not being able to study this has meant that we have been unable to say whether GPCRs are spatially organised and whether this affects cell signalling. If such spatial organisation exists, the question is also which mechanisms control it and how this influences the effects of drugs, for example,” says Dimitrios Stamou.
Creating 3D structures of cell membranes
Dimitrios Stamou and colleagues have developed a unique method for studying cell membranes.
The problem has been that researchers have attempted to study the cell membrane in 2D, and this has made studying the distribution of proteins in 3D impossible, since a membrane is not flat like a piece of paper, but instead meanders in peaks or valleys.
A normal microscope focuses on certain features of a cell membrane such as the peaks but not the rest. The higher the resolution, the greater the problem. This does not reveal how GPCRs are distributed.
The researchers in Dimitrios Stamou’s group have spent 4 years developing a method for combining 500 2D images of a membrane into a 3D image in which all the peaks and valleys in the image are razor sharp.
This also applies to all proteins on the surface – regardless of whether they are located on a peak or in a valley.
“With the method, we can not only see GPCRs but also other interesting proteins, such as H-RAS, which has an important role in treating cancer, and PIEZO1, which is important for sensing mechanical pressure, such as pressing two fingers together. This method clearly shows that the GPCRs are distributed with high concentrations in some locations and low concentrations in others,” explains Dimitrios Stamou.
GPCRs gather on the peaks of cell membranes
In the next phase of the study, the researchers investigated how the spatial organisation of the GPCRs is determined.
They gather on the peaks of the landscape that the cell membrane creates.
Although a cell membrane is fluid, and proteins and lipids flow in between each other, the concentration of GPCRs remains high at the peaks and correspondingly low in the valleys.
However, this results from the curve of the membrane and not its height.
If the curve is positive near a peak, this leads to enrichment of GPCRs, whereas a negative curve, such as in a valley, results in GPCR-depleted domains.
“The method we developed has thus enabled us not only to verify that GPCRs are spatially organised but also to identify the underlying molecular mechanism,” says first author Gabriele Kockelkoren, Center for Geometrically Engineered Cellular Systems, Department of Chemistry, University of Copenhagen.
Cell shape is more important than previously thought
According to Dimitrios Stamou, a cell’s shape affects the spatial organisation of GPCRs and thus also signalling into the cells, and this is crucial and enormously interesting because cell shapes vary.
For example, a neuron looks markedly different from a red blood cell or a kidney cell.
These shapes are extremely conserved in evolution and are similar for almost all living creatures on Earth.
The reason is probably that the shape of the cell strongly affects how GPCRs are spatially organised, which thus determines cell function.
“This opens up a new research field: investigating how changing the shape changes cell function. If function changes, what are the implications for treatment with a drug, for example? We can now begin to answer these types of questions,” explains Dimitrios Stamou.
Dimitrios Stamou and colleagues are now answering many of these questions and already have a large pipeline of scientific articles on the way.
This research shows that knowing the shape of a cell membrane enables the location of GPCRs to be predicted very precisely.
“In an extremely complicated environment in which proteins and lipids interact freely, having one property as dominant as this and being so decisive for how the cell acts is extraordinary. This is incredibly interesting and very unexpected,” concludes Dimitrios Stamou.