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A close look at individual membrane receptors

Membrane receptors are rather like the sensory organs of cells: they capture signals from the environment and transmit them into cells. The majority of research groups around the world working on membrane receptors are concentrating on the interactions between the receptors and the signalling molecules in the interior of cells and each individual receptor tends to be seen as a “black box”. The independent research group led by junior professor Dr. Maximilian Ulbrich of the BIOSS excellence cluster at the University of Freiburg has developed a high-resolution, real-time method for looking at individual membrane-bound receptors. What are membrane receptors composed of? Which proteins bind to them, and influence their dynamic activity? Questions such as these help elucidate the processes in synapses as well as in other tissues.

The independent group of researchers led by junior professor Dr. Maximilian Ulbrich (second from the left). © Dr. Maximilian Ulbrich

Ten years ago, it all seemed very simple: a signal docks to a cell and activates a receptor, which then communicates the message into the cell. Nowadays, it has become clear that receptors are complex machines composed of different subunits that interact with dozens of other proteins that modulate their function or transport them to specific sites in the membrane. “The processes that occur at the receptors of a cell are extremely complicated,” said junior professor Dr. Maximilian Ulbrich from the Centre for Biological Signalling Studies (BIOSS) and the Institute of Physiology at the Freiburg University Medical Centre. “Receptors are involved in complex molecule networks about whose composition and dynamic we know very little.” Ulbrich did his postdoctoral studies in Berkeley (California) from 2004 to 2009, during which time he developed a method for looking closely at individual receptors and their interaction partners. At present, Ulbrich and his three staff members are mainly interested in two receptor groups that play important physiological roles: G-protein coupled receptors (GPCRs) and ionotropic glutamate receptors.

Total reflection and the interior of receptors

With around one thousand members, GPCRs represent the largest family of cell surface receptors. They are found in all types of cells. GPCRs are integral membrane proteins that possess seven membrane-spanning domains. Typical GPCRs consist of either one or two subunits. The part of the molecule that is located inside the cell is coupled to a G-protein, which is a molecule that is able to interact with different signalling cascades. GPCRs are not firmly anchored in the membrane and somehow float in the lipid membrane. This is in contrast to the ionotropic glutamate receptors, which are synaptic receptors located primarily on the membranes of neuronal cells and enable signals to be transmitted into the cell. Ionotropic glutamate receptors are ion channels. They are firmly anchored at specific membrane sites and define the areas where electrical current can flow. When the neurotransmitter glutamate binds to these receptors, the ion channels open and lead to changes in the membrane potential, which is the basis for the transmission of signals in the nervous system.

The composition of proteins can be investigated by co-localising differently labelled subunits (here: green and red). In this photo, different subunits of the NMDA receptors were expressed together. Depending on the type of receptor, these subunits either always combine (left) or never interact (right). The single-molecule experiment clearly shows the results (the two microscope images at the top). © Dr. Maximilian Ulbrich

It is known that receptors are composed of different subunits with different characteristics. However, this knowledge is often virtually ignored in the investigation of signalling processes. Ulbrich and his team believe that much more research based on this knowledge needs to be done. “We believe that it is important to know whether a particular type of GPCR consists of two identical or two different subunits,” said Ulrich, who has also studied physics. “This can have decisive effects on receptor function.” In addition, the function of GPCRs and glutamate receptors can be modulated by keeping the channel open for longer or shorter periods of time. The period of time depends on the proteins that are bound to the receptors. So the question arises as to what happens in, at and around an individual receptor as it receives signals and translates them into cells. Ulbrich and his team of researchers use a method known as total internal reflection fluorescence (TIRF) to find answers.

Stoichiometry is key

Unfertilised eggs of the African clawed frog are illuminated with a laser (green beam) through the lens of a microscope. As the laser has a small angle of incidence, it is reflected on the surface of the glass and thus only illuminates the adherent membrane of the cell. When labelled with GFP, the individual molecules of the membrane proteins can be discerned as fluorescent spots. © Dr. Maximilian Ulbrich

At first sight, the method does not seem to be any different from classical microscopy methods. Ulbrich’s laboratory is equipped with a perfectly normal laser microscope. However, a closer look shows that the course of the light in the microscope is modified so that the light is reflected off the surface of a sample, rather than penetrating it. This is made possible by modifying the relatively flat incidence angle of the light beam. The lens and the camera attached to it thus capture light that has only streaked the surface of a specific cell contained in the sample under investigation. The cell itself remains in the dark; only the membrane is visible. In order to visualise the receptor molecules in the membrane, Ulbrich’s team labels the receptor molecules with fluorescent proteins such as GFP.

The sensitivity of modern CCD cameras enables the researchers to discern individual molecules. And since they are looking at living cells, they are even able to observe the movement of receptors and changes in their behaviour when exposed to a specific signal. In order to distinguish protein subunits from one another, the researchers use several different marker proteins that illuminate in different colours. They can therefore see which subunits are bound to each other and which are not.

“Our methods are best suited for analysing the stoichiometry of proteins,” said Ulbrich, pointing out that their work involves a lot of counting. In future, the Freiburg researchers hope to find out the proportion of different subunits in a specific GPCR type and whether this proportion is absolutely necessary for the function of a receptor. In another project, the researchers plan to measure how many modulatory proteins can bind to an AMPA-type glutamate receptor while it is exerting its function. Biochemical and genetic methods cannot be used to count the number of proteins as they only show whether, and not how many, modulatory proteins are present.


Own projects and cooperations

Ulbrich and his team also offer their know-how to other research groups. In a collaborative project with researchers from the USA, the researchers from Freiburg have been able to show that a receptor discovered by their American colleagues is composed of two subunits, at a ratio of 3:2. The team have since managed to perfect the method. “Now, we can focus on our own projects,” said Ulbrich adding that that they have chosen to work on GPCRs and glutamate receptors. In the near future, the Freiburg researchers plan to work on other topics of major interest. As part of BIOSS, they have links with scientists from many different research disciplines in Freiburg, including cell biology, neurobiology and immunobiology. Other scientists will be extremely keen to work with them, as membrane receptors play a key role in all known signalling networks, and hence in all areas covered by the life sciences.

Further information:
Junior Professor Dr. Maximilian Ulbrich
Habsburgerstr. 49
79104 Freiburg
Tel.: +49 (0)761/203 97183
Fax: +49 (0)761/203 97232
E-mail: max.ulbrich(at)physiologie.uni-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/a-close-look-at-individual-membrane-receptors