The transmembrane AMPA receptor regulatory protein gamma 4 is a more effective modulator of AMPA receptor function than stargazin (gamma 2).

AMPA receptors mediate the majority of the fast excitatory synaptic transmission in the brain. A family of recently described auxiliary proteins, the transmembrane AMPA receptor regulatory proteins (TARPs) gamma2, gamma3, gamma4, and gamma8, have been shown to modulate the trafficking of receptors to the plasma membrane as well as electrophysiological key properties. Most studies published to date focus exclusively on gamma2 (stargazin), neglecting the other three members of the TARP family. Here, we analyzed the modulation of electrophysiological properties of AMPA receptors by gamma4 and compare it with gamma2, using heterologous coexpression in human embryonic kidney 293 cells. We show for the first time that gamma4, a previously poorly examined TARP, modulates the desensitization properties of AMPA receptors significantly stronger than gamma2 does. In contrast, other properties such as kainate efficacy and current-voltage relationships are modulated in a similar way by both of these TARPs. From these TARP-specific effects, we propose an interaction mechanism between AMPA receptors and TARPs and address the physiological relevance of gamma4 and its regulatory effects, particularly on AMPA receptor desensitization properties, to developmental and regulatory processes in the brain.

Quantitative analysis of cotransfection efficiencies in studies of ionotropic glutamate receptor complexes.

Transient transfection of cultured mammalian cells is widely employed in the study of ionotropic glutamate receptors. Heteromeric expression is usually achieved by simultaneous transfection of various combinations of glutamate receptor subunit-encoding cDNAs. This approach is based on an "all-or-none" assumption, rarely verified experimentally, that any given cell expresses all subunits present during transfection. A similar assumption implicitly is made when cotransfection of a cDNA encoding a fluorescent marker protein is applied to distinguish transfected from untransfected cells. A further frequent assumption alleges that the ratio between cDNAs used in cotransfection experiments directs the assembly of receptor complexes in heterologous expression systems. To check the validity of these assumptions for ionotropic glutamate receptors as model transmembrane receptors, we generated fluorescently labeled receptor subunits and introduced them into HEK-293 cells by the calcium phosphate method. Analyzing the expression of multiple fusion proteins by confocal microscopy, we evaluated the coexpression efficiencies for various glutamate receptor cDNA combinations, cDNA amounts, and cDNA ratios. Several factors were found to influence the individual, cumulative, and cotransfection efficiencies, including the cDNA ratio, the nature of the expressed protein, and the specific combination of cotransfected cDNAs. After simultaneous transfection with equal amounts of several cDNAs, we demonstrate the consistent generation of several distinct populations of cells that express different receptor subunit combinations. The evidence we present suggests that cotransfected cells should always be independently tested for the expression of all target subunits before picking cells for the analysis of specific heteromeric receptor assemblies.

Functional significance of the kainate receptor GluR6(M836I) mutation that is linked to autism.

Previous studies revealed a linkage of the kainate receptor GluR6 with autism, a pervasive developmental disorder. Mutational screening in autistic patients disclosed the amino acid exchange M836I in a highly conserved domain of the cytoplasmic C-terminal region of GluR6. Here, we show that this mutation leads to GluR6 gain-of-function. By using the two-electrode voltage clamp technique we observed a significant increase of current amplitudes of mutant GluR6 compared to wild type GluR6. Western blotting of oocytes injected with mutant or wild type GluR6 cRNA and transfection of EGFP-tagged GluR6 receptors into COS-7 cells revealed an enhanced plasma membrane expression of GluR6(M836I) compared to wild type GluR6. Membrane expression of GluR6(M836I) but not of wild type GluR6 seems to be regulated by Rab11 as indicated by our finding that GluR6(M836I) but not wild type GluR6 showed increased current amplitudes and protein expression when coexpressed with Rab11. Furthermore, injection of GTP plus Rab11A protein into oocytes increased current amplitudes in GluR6(M836I) but not in wild type GluR6. By contrast, Rab5 downregulated the currents in oocytes expressing wild type GluR6 but had only little, statistically not significant effects on currents in oocytes expressing GluR6(M836I). Our data on altered functional properties of GluR6(M836I) provide a functional basis for the postulated linkage of GluR6 to autism. Furthermore, we identified new mechanisms determining the plasma membrane abundance of wild type GluR6 and GluR6(M836I).

Cellular retinoic acid-binding protein 2 is down-regulated in prostate cancer.

Prostate cancer is among the most frequent tumours in industrialized nations and many questions remain open concerning the molecular events underlying its development and progression. In the present study we have combined cDNA array hybridization to laser-assisted microdissection (LAM) in order to investigate differences in gene expression between epithelial and stromal cells of prostate cancer and normal peripheral prostate tissue. Results have been verified for selected candidate genes by quantitative real-time RT-PCR. Using this approach and immunohistochemistry we could demonstrate a down-regulation of cellular retinoic acid binding protein 2 (CRABP2) mRNA and protein in carcinoma cells compared to normal glandular cells. CRABP2 is a main regulator of anti-carcinogenic activities of retinoic acid and may become a novel diagnostic marker and experimental therapeutic tool for prostate cancer. In addition, results of cDNA array hybridization suggest an up-regulation of 34 further genes and a down-regulation of 6 genes in cancer tissues compared to normal peripheral prostate tissues. Several of these genes have already been reported to be associated with carcinogenesis in organs such as the prostate.

A novel component of the ubiquitin pathway, ubiquitin carboxyl extension protein 1 is overexpressed in prostate cancer.

Prostate cancer is among the most common tumors in industrialized nations. However, little is known about the molecular events underlying its development. In the present study we used suppressive subtraction hybridization (SSH) in combination with laser-assisted microdissection in order to compare gene expression between prostate carcinoma and the normal prostate proper. Both are mixed tissues which consist of an epithelial and a stromal compartment. We first compared mRNA (cDNA) expression by SSH and then used real-time quantitative RT-PCR analysis of microdissected tissue probes in order to verify differential expression of subtracted cDNA clones. We also used differentially expressed cDNAs for the synthesis of radiolabelled riboprobes in order to attribute differential expression to specific cell types in tissue sections by in situ hybridization. Using this approach we found an up-regulation of ubiquitin carboxyl extension protein 1 (UBCEP-1) mRNA in prostate carcinoma cells compared to the normal glandular epithelium of the prostate proper. UBCEP-1 mediated ubiquitin chain elongation may promote prostate carcinoma development by increasing via the proteasome pathway the degradation of proteins which are involved in growth inhibition or apoptosis.

Analysis of microdissected prostate tissue with ProteinChip arrays--a way to new insights into carcinogenesis and to diagnostic tools.

Prostate carcinomas are one of the most common malignancies in western societies. The pathogenesis of this tumor is still poorly understood. These tumors present with two characteristic features: epithelial-mesenchymal interactions, which play a pivotal role for tumor development and most of clinically manifest cancers arise in prostate proper compared to a minority of tumors which develop in the transitional zone. Deciphering the epithelial-mesenchymal cross talk and identification of molecular pecularities of the sub-populations of cells in different zones can therefore help understanding carcinogenesis and development of new, non-invasive tools for the diagnosis and prognosis of prostate carcinomas which has remained a challenge until today. A ProteinChip array technology (SELDI = surface enhanced laser desorption ionization) has been developed recently by Ciphergen Biosystems enabling analysis and profiling of complex protein mixtures from a few cells. This study describes the analysis of approximately 500-1000 freshly obtained prostate cells by SELDI-TOF-MS (surface enhanced laser desorption ionization time-of-flight mass spectrometry). Pure cell populations of stroma, epithelium and tumor cells were selected by laser assisted microdissection. Multiple specific protein patterns were reproducibly detected in the range from 1.5 to 30 kDa in 28 sub-populations of 4 tumorous prostates and 1 control. A specific 4.3 kDa peak was increased in the prostate tumor stroma compared to normal prostate proper and transitional zone stroma and increased in prostate tumor glands compared to normal prostate proper and transitional zone glands. Coupling laser assisted microdissection with SELDI provides tremendous opportunities to identify cell and tumor specific proteins to understand molecular events underlying prostate carcinoma development. It underlines the vast potential of this technology to better understand pathogenesis and identify potential candidates for new specific biomarkers in general which could help to screen for and distinguish disease entities, i.e. between clinically significant and insignificant carcinomas of the prostate.