The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.

Tim23, the central subunit of the TIM23 protein-translocation complex, forms a voltage-gated channel in the mitochondrial inner membrane (MIM), an energy-conserving membrane that generates a proton-motive force to drive vital processes. Using high-resolution fluorescence mapping of a channel-facing transmembrane segment (TMS2) of Tim23 from Saccharomyces cerevisiae, we demonstrate that changes in the energized state of the MIM cause marked structural alterations in the channel region. In an energized membrane, TMS2 forms a continuous α-helix that is inaccessible to the aqueous intermembrane space (IMS). Upon depolarization, the helical periodicity of TMS2 is disrupted, and the channel becomes exposed to the IMS. Kinetic measurements confirm that changes in TMS2 conformation coincide with depolarization. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins coupled to processes such as channel gating.

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous G protein-coupled receptors (GPCRs) such as rhodopsin and the beta-adrenergic receptor exhibit unique structural and functional characteristics. Computer models also predict this structure for the cannabinoid CB2 receptor, another member of the GPCR superfamily. In our study, a peptide corresponding to helix 8 of the CB2 receptor was synthesized chemically and its secondary structure determined by circular dichroism (CD) and (1)H NMR spectroscopy. NMR and CD revealed an alpha-helical structure in this region in both dodecylphosphocholine micelles and dimethylsulfoxide, in contrast to a random coil configuration found in aqueous solvent. This finding is in good agreement with other previous GPCR structural studies including X-ray crystallography. By combining our finding with other studies, we further hypothesize that the amphipathic nature of helix 8 can play a significant role in the function and regulation of CB receptors as well as other GPCRs in general.

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Abstract.A major goal of treatment strategies for cancer is the development of agents which can block primary tumor growth and development as well as the progression of tumor metastasis without any treatment associated side effects. Using mini peptide display (MPD) technology, we generated peptides that can bind to the human vascular endothelial growth factor (VEGF) receptor KDR. These peptides were evaluated for their ability to block angiogenesis, tumor growth and metastasis in vitro and in vivo. A D-amino acid peptide with high serum stability (ST100,059) was found to have the most potent activity in vitro as indicated by inhibition of VEGF stimulation of endothelial cells. It was also found to be the most active of the series in blocking VEGF-mediated activity in vivo, as measured in Matrigel-filled angioreactors implanted in mice. ST100,059 blocks VEGF-induced MAPK phosphorylation, as well as inhibits VEGF-induced changes in gene expression in HUVEC cells. In in vivo studies, treatment of female C57BL/6 mice inoculated with B16 mouse melanoma cells with ST100,059 resulted in a dose-dependent decrease in tumor volume and lung metastasis as compared to control groups of animals receiving vehicle alone. These studies demonstrate that by using MPD, peptides can be identified with enhanced affinity relative to those discovered using phage display. Based on these studies we have identified one such peptide ST100,059 which can effectively block tumor growth and metastasis due to its anti-angiogenic effects and ability to block intracellular signaling pathways involved in tumor progression.

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