Purification and functional reconstitution of the rat organic cation transporter OCT1.

The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.

The MAM (meprin/A5-protein/PTPmu) domain is a homophilic binding site promoting the lateral dimerization of receptor-like protein-tyrosine phosphatase mu.

The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTPmu belongs to the MAM-containing subclass of protein-tyrosine phosphatases (PTP) able to promote cell-to-cell adhesion. Here we provide experimental evidence that the MAM domain is a homophilic binding site of PTPmu. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTPmu ectodomain at the cell surface. The presence of two disulfide bridges in the MAM molecule was evidenced and their integrity was found to be essential for MAM homophilic interaction. Our data also indicate that PTPmu ectodomain forms oligomers and mediates the cellular adhesion, even in the absence of MAM domain homophilic binding. Reciprocally, MAM is able to interact homophilically in the absence of ectodomain trans binding. The MAM domain therefore contains independent cis and trans interaction sites and we predict that its main role is to promote lateral dimerization of PTPmu at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.

Comparative analysis of high-affinity ligand binding and G protein coupling of the human CXCR1 chemokine receptor and of a CXCR1-Galpha fusion protein after heterologous production in baculovirus-infected insect cells.

In order to perform biochemical and pharmacological characterization of CXCR1, we designed several CXCR1 constructs. All constructs, including a CXCR1-G(i2)alpha fusion protein, were produced in insect cells after infection with recombinant baculovirus. The recombinant receptors exhibited specific high-affinity binding of (125)I-labelled interleukin-8, and Scatchard transformation of the binding data indicated the presence of a population of single homogenous binding sites. Furthermore, the pharmacological profiles for the different CXCR1 constructs produced in the baculovirus-infected insect cells were almost identical to those reported for CXCR1 on human neutrophils. Interestingly, when the CXCR1 constructs were coproduced with G(i2) protein as a result of coinfection with baculoviruses encoding the G(i2)alpha-, the beta- and the gamma- subunits, the B(max) values were significantly increased. Hence, the level of FlagCXCR1Bio, after coproduction with G(i2) protein, was found to be almost 10 times higher than that of the FlagCXCR1Bio alone. However, no differences in the K(i) values were observed of the receptor constructs produced either after single infection or coinfection of insect cells. The addition of guanyl-5'-yl imidodiphosphate resulted in a dramatic reduction of the number of binding sites; however, the K(i) values remained unchanged, indicating coupling of the receptor to the guanine nucleotide-binding protein.

Production of the human D2S receptor in the methylotrophic yeast P. pastoris.

In order to evaluate the methylotrophic yeast Pichia pastoris as means for high-yield production of homogenous D(2S) receptor protein, we have expressed the unmodified D(2S) receptor and various D(2S) receptor fusion constructs under the transcriptional control of the highly inducible promotor of the P. pastoris alcoholoxidase 1 gene in strain SMD1163. Fusion of the D(2S) receptor gene to the alpha-factor preprosequence proved to be essential for receptor production. For the receptor fusion constructs a gene dosage of more than two copies per cell increased production levels three- to sixfold. Adding various dopaminergic ligands to the induction medium increased yields up to tenfold, reaching 51,500 +/- 5700 receptors/cell. Immunoblot analysis of the effect of tunicamycin on D(2S) receptor fusion proteins and immunoprecipitation of metabolically labeled wild-type and glycosylation-deficient D(2S) receptor fusion proteins revealed that the high-mannose-type glycosylation of the D(2S) receptor prevents cleavage of the alpha-factor prosequence by the Kex2 endopeptidase. Abolishing glycosylation restored correct processing. Immunogold electron microscopy showed that recombinant yeast cells overproducing the D(2S) receptor developed membrane stacks harboring the receptor protein. The pharmacological profile of the recombinant D(2S) receptor was similar to that reported for neuronal D(2) receptors independent of glycosylation and processing. In conclusion, the D(2S) receptor can readily be produced in P. pastoris with high yield suitable for receptor purification and future structural studies.

Impact of aromatic residues within transmembrane helix 6 of the human gonadotropin-releasing hormone receptor upon agonist and antagonist binding.

To investigate the impact of aromatic residues within transmembrane helix 6 (TMH6) of the human gonadotropin-releasing hormone receptor (GnRH-R) on agonist and antagonist binding, residues Y(283), Y(284), W(289), Y(290), W(291), and F(292) were exchanged to alanine and analyzed comprehensively in functional reporter gene and ligand binding assays. Whereas receptor mutants Y(283)A, Y(284)A, and W(291)A were capable of neither ligand binding nor signal transduction, mutants W(289)A, Y(290)A, and F(292)A were functional: the F(292)A mutant behaved like wild-type receptor, while mutants W(289)A and Y(290)A differentiated between agonistic and antagonistic ligands. On the basis of the high-resolution X-ray structure of bovine rhodopsin as well as available data on GnRH-R mutants, models for ligand-receptor interactions are proposed. The model for D-Trp(6)-GnRH (Triptorelin) binding, representing a superagonistic ligand, is in full accordance to available data. Furthermore, new interactions are proposed: pGlu(1) interacts with N(212) in transmembrane helix 5, Tyr(5) with Y(290), and D-Trp(6) with W(289). The binding behavior of mutants W(289)A and Y(290)A corresponds to the proposed binding model for the antagonist Cetrorelix. In summary, our data as presented indicate that Y(290) plays a key function in agonist but not antagonist binding.