Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties, mAbs, 5:1, 22-33,

The N-terminal extracellular parts of human G-protein coupled receptor class B, for example, receptors for secretin, glucagon, or parathyroid hormone, are involved in ligand binding. To obtain structural and functional information on the N-terminal receptor fragment of human parathyroid hormone receptor 1 (PTHR1), the truncated receptor was expressed in the cytosol of Escherichia coli in the form of inclusion bodies. Oxidative refolding of inclusion body material resulted in stable, soluble, monomeric protein. Ligand binding was proved by surface plasmon resonance spectroscopy and isothermal titration calorimetry. Refolded receptor fragment was able to bind parathyroid hormone with an apparent dissociation constant of 3-5 microM. Far-UV circular dichroism spectra showed that the refolded polypeptide contained approximately 25% alpha-helical and 23% beta-sheet secondary structures. Analysis of the disulfide bond pattern of the refolded receptor fragment revealed disulfide bonds between Cys170 and Cys131, Cys148 and Cys108, and Cys117 and Cys48. These results demonstrate that the extracellular N-terminal domain of the parathyroid hormone receptor (PTHR1) possesses a well-defined, stable conformation, which shows a significant ligand binding activity.

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-␤, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-␤, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-␣-dependent import of proteins with a classical NLS.Ran/TC4 is a highly abundant, small GTP-binding and -hydrolyzing protein that is located predominantly in the nucleus (8,12). Ran has the biochemical properties of a GTPase switch cycling between two conformational states, the GTP-bound state and the GDP-bound state. The intrinsic rates of nucleotide exchange and GTP hydrolysis are very low and can be increased up to five orders of magnitude by the regulatory proteins RCC1 and RanGAP1, respectively (4,8,22). The first indications concerning the functions of Ran came from the analysis of mutants of these Ran regulators in both mammalian and yeast cells. These studies implicated Ran in a variety of processes, including the onset of mitosis, initiation of S phase, exit from mitosis, maintenance of nuclear structure, and premRNA processing and mRNA export into the cytoplasm (for reviews, see references 41 and 43).Furthermore, in vitro studies with permeabilized cells showed that Ran is an essential factor for the nuclear localization signal (NLS)-dependent nuclear protein import (30, 31). Macromolecular transport across the nuclear envelope occurs at the nuclear pore complexes (NPCs) and involves the import of proteins into the cell nucleus and the export of RNAs and proteins. Four soluble cytosolic factors are required to reconstitute nuclear import of NLS substrates in vitro. In addition to Ran, these are importin-␣ and importin-␤ (also known as karyopherin-␣ and karyopherin-␤) (17,19,36) and NTF2 (alternatively known as pp15 or p10) (32, 34). Together, importin-␣ and importin-␤ comprise the import receptor complex, where importin-␣ binds proteins bearing an NLS and importin-␤ mediates the interaction with the NPC. Ran appears to be required for at least two steps in nuclear import. First, translocation through the nuclear pore requires GTP hydrolysis by Ran, probably even as the sole source of energy (47). Second, the disassembly of the importin-␣/-␤-NLS protein complex following translocation ...