Ellen I. Closs, Ph.D.

• Short CV
• Research achievments

Research Achievements:

As a PhD student in the department of Molecular Pathology of the GSF research center in Neuherberg (head: Professor Volker Erfle), I investigated the influence of retroviral infection on the differentiation and transformation of chondroblasts and osteoblasts in organ cultures. These studies demonstrated that differentiated chondrocytes can trans-differentiate into osteoblasts and that the transscription factor c-fos plays a crucial role in the trans-differentiation process.

After obtaining my PhD from the Ludwig-Maximilians University in Munich in 1989, I was granted a two years scholarship from the Deutsche Forschungsgemeinschaft. With that, I joined Professor James Cunninghams laboratory at the Harvard Medical School in Boston , USA , to search for the cellular function of a virus receptor that had been identified by his group.  These studies led to the identification of the first mammalian amino acid transporter (mCAT-1, for murine cationic amino acid transporter).  Subsequently, we discovered two proteins related to mCAT-1, (mCAT-2A and mCAT-2B) that also function as transport proteins for cationic amino acids, but differ in their transport properties and tissue distribution.

In 1993, I moved back to Germany to lead an independent research group in the department of Pharmacology in Mainz (head: Professor Ulrich Förstermann).  Here, we identified and characterised the human CAT isoforms hCAT-1, -2A, -2B and -3 by expression in Xenopus laevis oocytes. In collaboration with Professor Hermann Nawrath in our department, we investigated the voltage-dependence of L‑arginine transport by all established hCATs.  We also studied the putative family member SLC7A4 or hCAT-4. It’s expression in the plasma membrane of X. laevis oocytes or mammalian cells was not sufficient to mediate amino acid transport, suggesting that it needs to interact with accessory proteins to form an active transporter.  Expression of the individual CAT isoforms is cell type dependent and highly regulated.  We could demonstrate that changes in CAT-1 mRNA expression are not necessarily reflected in changes in protein expression and unravel part of the complex posttranscriptional regulation of CAT expression.  In addition, we found that the cell surface expression (and thus the activity) of the CAT proteins can be regulated independent of the overall protein expression, for example by activation of classical PKC leading to a reduction in transporter protein expressed in the plasma membrane. When analyzing the structure/function relationship of the CAT proteins, we detected a protein domain that determines the transport properties of the CAT proteins and used site directed mutagenesis to identify crucial amino acid residues determining the apparent substrate affinity.

The second main research focuses in my group concerned the role of plasma membrane transport and intracellular arginine sources for substrate supply of nitric oxide synthases (NOS) and other arginine-dependent enzymes. Using RFL-6 reporter cell assays and HPLC analysis, we demonstrated that endothelial cells have an L-arginine pool that is not freely exchangeable with the extracellular space and that the endothelial NOS (eNOS) in these cells has access to this pool. We further established that this intracellular arginine pool consists of two independent substrate sources: recycling of citrulline to arginine and protein breakdown. Towards the pharmacokinetic of NOS inhibitors, we showed that the CAT proteins do also transport some NOS inhibitors, but that there are differences in the substrate recognition by NOS and CATs. We could establish that CAT-2B is often co-induced with the inducible NOS (iNOS) suggesting that CAT-2B has an important role in supplying substrate for NO synthesis by this NOS isoform.  However, the signal transduction pathways regulating CAT-2B and iNOS expression are not identical.

Other research activities in my lab have included other transport proteins (MRP4, OCTN2), soluble guanylyl cyclase (the major effector molecule of NO), and BH4 (an essential co-factor of NOS).