Journal of receptor research最新文献

Arginine vasopressin (AVP) binds to two distinct receptors to initiate vasopressor (V1 receptor) and hydroosmotic actions (V2 receptor). Internalization and recycling of the V1 receptor in cultured vascular smooth muscle cells and hepatocytes have recently been demonstrated. However, the receptor cycle of the AVP V2 receptor in the renal collecting tubules has not yet been well defined. Therefore, the present study was undertaken to investigate the AVP V2 receptor cycle, including AVP binding to the surface receptor, internalization and potential recycling in isolated outer medullary collecting tubules. The maximal AVP surface binding was reached in 10 min, and 25 micrograms/ml trypsin completely inhibited the surface binding. A Scatchard plot of 125I-AVP surface binding indicated a single population of V2 receptors with a Kd of 1.92 x 10(-9) M and a Bmax of 1.77 x 10(-11) M or 590 fmoles/mg protein. 81.7% (72-85%) of specific bound receptor was internalized (specific surface binding: 742.8 +/- 111.1 vs internalized binding: 607.3 +/- 27.8 fmoles bound/mg protein). More than 90% of surface bound receptor was recycled to the cell surface after internalization (control surface binding: 584.0 +/- 64.0 vs recycled surface binding: 546.6 +/- 32.0 fmoles bound/mg protein). Cycloheximide (40 micrograms/ml) did not inhibit the receptor recycling (control recycled surface binding: 546.6 +/- 32.0 vs cycloheximide recycled surface binding: 505.0 +/- 54.8 fmoles bound/mg protein), thus suggesting that the receptors were not resynthesized after dissociation from the receptor-ligand complex. These studies therefore demonstrate that the AVP V2 receptor is internalized and recycled in the rat renal collecting tubule.

The alpha 1-adrenoceptor subtypes in rat lung were characterized according to their binding of [3H]-prazosin or [3H]-WB4101 and were compared with that in rat liver. [3H]-prazosin bound with high affinity to an apparently homogeneous population of sites in rat lung. The binding of [3H]-prazosin was inhibited by WB4101, benoxathian and 5-methylurapidil biphasically but the proportions differed between WB4101 or benoxathian and 5-methylurapidil. In the lung membranes pretreated with chloroethylclonidine a single population with high affinity for WB4101 and benoxathian was detected while 5-methylurapidil still discriminated two sites of distinctly different affinities. These results suggest that the WB4101-high affinity sites of rat lung were subdivided further into two subclasses according to 5-methylurapidil binding affinity. In fact, [3H]-WB4101 bound to lung membranes with two different affinities and the high affinity binding sites were subdivided by 5-methylurapidil into two classes. By contrast, [3H]-prazosin or [3H]-WB4101 binding sites of liver membranes were detected as a single population with high affinity for prazosin but with low affinity for WB4101, benoxathian and 5-methylurapidil. These results suggest that the alpha 1-adrenoceptors of rat lung are composed of three distinct subtypes (alpha 1A, alpha 1B and unknown subtypes) while that of liver is of alpha 1B subtype. Two radioligands with different affinities may be used as powerful probes to identify receptor subclasses.

Previous studies have revealed two size classes of alpha 1b-adrenergic receptor mRNAs, 3.3 kb and 2.7 kb, in the Sprague Dawley rat that are transcribed from a single gene and are expressed in approximately equal amounts in liver. Only the 2.7 kb mRNA is expressed in heart. Both alpha 1b-adrenergic receptor mRNAs appear to share extensive regions of homology, therefore, we used oligonucleotide-directed ribonuclease H mapping to detect sequence differences between the two transcripts. Initial experiments using oligo (dT)-directed RNase H hydrolysis indicated that the two mRNAs have poly [A+] tails of identical length. By using region-specific cDNA probes, we determined that the sequence difference between the two alpha 1b-adrenergic receptor mRNAs lies in the 5' end, upstream from the known initiator AUG in the 2.7 kb transcript. In addition, results from ribonuclease protection assays and Northern blot analysis in which an oligonucleotide was used as the probe suggested that both alpha 1b-adrenergic receptor mRNAs are transcribed from the same DNA strand.

We previously demonstrated that, in addition to the estrogen receptor, the Antiestrogen Binding Site (ABS) is also a potent mediator of the antitumorous activity of the clinical drug tamoxifen. Because of report discrepancies in the binding parameters of rat liver ABS we first attempted to improve binding study conditions. In this way buffer, protein concentration, methodology for bound/free ligand separation and phospholipidic ratio were determined. This work was used to evaluate the Stoke radius (4.4 S) and isoelectric point (pH = 6.6) of the protein in its native state. These studies constituted the obligatory transition from rat liver to pure ABS protein.

Our knowledge of the biological role of the antiestrogen binding site ABS in the antitumoral activity of tamoxifen, will be increased with the determination of its coding gene sequence. To this end our team has for some time attempted to purify this membranous protein. In this work we report the purification to homogeneity of ABS from rat liver in a six step succession. Specific photolabeling with a tritiated photoprobe, solubilization of rat liver microsomes, chromatofocusing of the labeled proteins, preparative electrophoresis on polyacrylamide gel, and two consecutive high performance liquid chromatography separations on C4 hydrophobic resin produced 2.5 micrograms of pure ABS by silver stain analysis of SDS-PAGE. The NH2-terminal residue of the protein appears to be blocked, which hinders the Edman degradation method for obtention of the whole protein sequence.

We previously reported that dopamine (DA) inhibited the release of human placental lactogen (hPL) from human placental cells. We also demonstrated the presence of D2-dopamine receptors in membrane preparations of human term placenta. The aim of the present study was to characterize these D2 receptors on freshly isolated human trophoblastic cells. The binding of [3H]-spiperone to these cells showed a curvilinear Scatchard plot suggesting the presence of two classes of binding sites (Kd1 = 1.26nM; Kd2 = 44.3nM). Competition experiments showed the following inhibitory binding potencies: serotonin-2 (5-HT2) > or = D2 >>> alpha-adrenergic, beta-adrenergic, D1-dopamine, thus suggesting the presence of 5-HT2 binding sites. We have examined this possibility by blocking [3H]-spiperone binding to 5-HT2 receptors in the presence of 50nM ketanserin, a selective antagonist of 5-HT2 sites. Under this condition, the linear Scatchard plot obtained suggested a single population of homogeneous binding sites for [3H]-spiperone with a Kd of 0.55nM. To further characterize placental D2 receptors we conducted binding experiments with [3H]-raclopride, an more selective D2 antagonist. The linear Scatchard plot obtained with this ligand suggested one class of binding sites for [3H]-raclopride (Kd = 6nM) with the following inhibitory potencies: D2 >>> beta-adrenergic >> 5-HT2, D1, alpha-adrenergic. These results suggest an important paracrine function for DA in human placenta and show for the first time that [3H]-spiperone binds putative 5-HT2 receptors in human placenta.

HeLa cells express low levels of beta-adrenergic receptor (beta AR) of the beta 2-subtype. When exposed to sodium butyrate, receptor levels increased up to 4-fold in a time dependent manner, reaching a maximum after 12 to 15 h of treatment. Sodium butyrate treatment also caused a 3 to 4 fold increase in levels of beta 2AR mRNA determined by hybridization blot analysis. The induction of beta 2AR mRNA temporally preceded the increase in receptor binding activity, reaching a maximum after 4 to 6 h of treatment, and remaining elevated for up to 24 h. Prior exposure of the cells to the protein synthesis inhibitor cycloheximide prevented the butyrate-induced increase in receptor binding but had no effect on the increase in receptor mRNA. Blocking DNA synthesis and cell growth by excess thymidine did not increase beta 2AR mRNA or binding or prevent the effects of sodium butyrate. Thus, butyrate appears to induce beta 2AR mRNA by a mechanism independent of DNA and protein synthesis.

We treated human neutrophils with papain to remove the external 2-kDa domain and along with it the two oligosaccharide side chains of the N-formyl peptide chemotactic receptor and investigated what effect their absence has on the ligand-receptor complex internalization. After prelabeling of the cells with 125I-hexapeptide for 5 min at 22 degrees C, about 95% of the bound radioactivity was located on the cell surface. During the first 5-min incubation at 37 degrees C both the control and papain-treated cells internalized 73% of the receptor-ligand complexes suggesting that internalization is very rapid in human neutrophils and that removal of the external domain and the carbohydrates of the receptor does not affect the rate. However, the truncated receptor-ligand complexes were degraded at a faster rate because the radioactivity released into the medium was significantly higher and correspondingly the acid-resistant radioactivity significantly lower in the papain-treated neutrophils than in control cells already at 5 min and all subsequent time points. The radioactivity accumulated in the medium of the control and papain-treated neutrophils represented inactivated 125I-hexapeptide as less than 5% of it at 5, 30 and 120 min were capable of rebinding. No receptor recycling was detected in either cells. These results indicate that removal of the 2-kDa external domain and the carbohydrates of the N-formyl chemotactic receptors has little effect on the internalization rate of the receptor-ligand complexes but accelerates markedly their intracellular degradation.

The human neurokinin-1 receptor has been expressed in insect cells using a recombinant baculovirus. The expression level is about 10 times higher than that obtained in mammalian cells. The recombinant receptor was solubilized with CHAPS, and a PEG precipitation procedure was shown to be effective in regaining high affinity substance P binding. This system should allow large scale purification of the human neurokinin-1 receptor.