Protein Trafficking (Ralf Schülein)

Research

1. Functional significance of cleavable signal peptides of GPCRs

Over the years, we could show that signal peptides of GPCRs serve surprisingly different functions in addition to their role in mediating ER targeting/integration. They may facilitate N-tail translocation of the receptors across the ER membrane or increase receptor expression at the plasma membrane. Sequences following the signal peptide may also assist in translocon gating. Of particular interest are the signal peptides of the corticotropin-releasing factor (CRF) receptors. These receptors are involved in the body’s responses to stress and play an important role in the development of anxiety and depression disorders. We could show that the corticotro­pin-releasing factor receptor type 2a (CRF2(a)R), possesses a unique N-terminal pseudo signal peptide which is uncleaved and forms an additional hydrophobic domain at the N tail of the receptor. We have studied the functional significance of this novel GPCR domain in comparison to the conventional cleaved signal peptide of the homologous corticotropin-releasing factor type 1 (CRF1R). We could show that the presence of the pseudo signal peptide prevents receptor dimerization and, as a consequence, prevents coupling of the receptor to the Gi protein. The CRF2(a)R is thus exclusively expressed as a monomer and is only able to couple to Gs whereas the CRF1R is expressed as a dimer and is able to couple to both, Gs and Gi (Fig. 2). In the future, we would like to express CRF receptor monomers and dimers in live animals to analyze their functional significance for the body’s responses to stress. These results may also shed new light on the development of anxiety and depression disorders.

2. Development of novel biosynthesis inhibitors for specific GPCRs and identification of general inhibitors of the translocon complex

For a pharmacological application, two types of inhibitors acting at the level of the translocon complex may be important in the future: Inhibitors interfering with signal sequence functions (type 1 inhibitors) could block the biosynthesis of specific GPCRs and other proteins and consequently represent an alternative to conventional antagonistic drugs acting at the level of the mature proteins. On the other hand, general blockers of the translocon complex may represent novel tumour drugs (type 2 inhibitors).

The cyclodepsipeptide cotransin was described as an inhibitor at the level of the translocon complex that acts in a signal sequence-discriminatory manner. Originally, cotransin was shown to inhibit the biosynthesis of only a small subset of proteins (mixed type 1/type 2 inhibitor). However, actual cotransin selectivity, was unknown, nor was it known which properties of a signal sequence are responsible for its cotransin sensitivity. To address these questions, we performed a proteomic study (cooperation with the Mass Spectrometry group of the FMP) and found that at saturating concentrations, the biosynthesis of almost all secreted proteins was cotransin-sensitive. In contrast, the biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Moreover, we were able to identify the first conformational consensus motif in signal anchor sequences mediating cotransin sensitivity (see figure below).

 

Although cotransin has a very interesting mechanism of action, its pharmacological application seems to be precluded due to its mixed type 1/type 2 inhibitor properties. We therefore set up a sequential high troughput screening assay to identify pure type 1 inhibitors for the signal peptide of the CRF1R and type 2 inhibitors for the translocon complex in general (cooperation with the Screening Unit and the Medicinal chemistry group of the FMP) of the FMP). Using this assay and the compound library of the FMP, we could identify a novel type 2 inhibitor acting at the level of the translocon complex (compound FMP-401319-3). The substance interferes with a step after the targeting and transfer of the nascent chains to the translocon, but before the growing peptide is processed through this complex.

3. Influence of cell aging processes on GPCR trafficking and signalling.

Cell aging is usually accompanied by the formation of protein aggregates in cells. The presence of these aggregates leads to a progressive sequestration of cellular proteins which are normally engaged in protein folding such as molecular chaperones. The effects of these cell aging processes on GPCR trafficking and signalling are largely unknown. We have recently started a new project addressing these questions (cooperation with the Proteostasis in Aging and Disease group of the FMP). 

Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

Like many sites, we use cookies to optimize the user's browsing experience. Data Protection OK