Covalent cross-linking of growth hormone-releasing factor to pituitary receptors.

We have used a bifunctional cross-linker, disuccinimidyl suberate, to covalently attach [125I]human pancreatic GH-releasing factor (GHRF) (-1-40)OH to bovine pituitary membranes and rat anterior pituitary cells. Covalently radiolabeled membrane and cell preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. In the former case, we observed the specific labeling of a polypeptide with an apparent mol wt of 75,000 +/- 3,000. The labeling of this species was specific for GHRF, as evidenced by the fact that it was inhibited in a dose-dependent fashion with increasing concentration of unlabeled GHRF. Furthermore, the radiolabeling was inhibited in the presence of excess unlabeled GHRF analogs but not unrelated peptides such as insulin and rat GH. The size of the radiolabeled band was the same in both bovine pituitary membranes and rat anterior pituitary cells. The extent of radiolabeling was dependent on the amount of membrane or the number of cells present during the binding reaction. These observations indicate that the mol wt 75,000 species is a ligand-binding subunit of the GHRF receptor in the pituitary. Under nonreducing conditions, a species much larger than mol wt 200,000 was specifically radiolabeled, again in both bovine pituitary membranes and rat cells. This result suggests the possibility that the ligand-binding subunit might be disulfide-linked to other subunit(s) forming homo- and heterooligomers.

Molecular and functional characterization of recombinant human metabotropic glutamate receptor subtype 5.

We have isolated and characterized overlapping cDNAs that encode two isoforms of the human metabotropic glutamate receptor subtype 5 (hmGluR5). The deduced amino acid sequences of human and rat mGluR5a are 94.5% identical. However, a region in the putative cytoplasmic domain (SER926-ALA1121) displays significant sequence divergence. Genomic analysis of this region showed that the sequence divergence results from species-specific differences in the genomic sequences, not from alternative splicing. The distribution of mGluR5 mRNA in human brain was most strongly detected throughout the hippocampus, with moderate levels in the caudate-putamen, cerebral cortex, thalamus, and deep cerebellar nuclei, and at low levels in the cerebellar cortex. Activation of both hmGluR5a and hmGluR5b transiently expressed in Xenopus oocytes and HEK293 cells was coupled to inositol phosphate (InsP) formation and elevation of the intracellular free calcium ([Ca2+]i). The agonist rank order of potency for activating recombinant hmGluR5a receptors in either system was quisqualate > L-glutamate > 1S,3R-ACPD. Both the quisqualate stimulated InsP and [Ca2+]i were inhibited by (+)-MCPG. Recombinant human mGluR5a was also stably expressed in mouse fibroblast Ltk- cells, in which the efficacy and potency of quisqualate were unchanged for more than 30 cell passages.

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside.

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.