Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel.

N-type calcium channels are omega-conotoxin (omega-CgTx)-sensitive, voltage-dependent ion channels involved in the control of neurotransmitter release from neurons. Multiple subtypes of voltage-dependent calcium channel complexes exist, and it is the alpha 1 subunit of the complex that forms the pore through which calcium enters the cell. The primary structures of human neuronal calcium channel alpha 1B subunits were deduced by the characterization of overlapping complementary DNAs. Two forms (alpha 1B-1 and alpha 1B-2) were identified in human neuroblastoma (IMR32) cells and in the central nervous system, but not in skeletal muscle or aorta tissues. The alpha 1B-1 subunit directs the recombinant expression of N-type calcium channel activity when it is transiently co-expressed with human neuronal beta 2 and alpha 2b subunits in mammalian HEK293 cells. The recombinant channel was irreversibly blocked by omega-CgTx but was insensitive to dihydropyridines. The alpha 1B-1 alpha 2b beta 2-transfected cells displayed a single class of saturable, high-affinity (dissociation constant = 55 pM) omega-CgTx binding sites. Co-expression of the beta 2 subunit was necessary for N-type channel activity, whereas the alpha 2b subunit appeared to modulate the expression of the channel. The heterogeneity of alpha 1B subunits, along with the heterogeneity of alpha 2 and beta subunits, is consistent with multiple, biophysically distinct N-type calcium channels.

Characterization of growth hormone releasing factor analog expression in Saccharomyces cerevisiae.

An analog of growth hormone releasing factor (GRF), [Leu27]GRF(1-40)-OH, has been expressed and secreted in Saccharomyces cerevisiae under the control of the alpha-factor gene promoter and prepro sequence. A single pair of consecutive basic residues served as a processing site between the alpha-factor sequences and the GRF sequences. [Leu27]GRF(1-40)-OH from fermentor broth containing 20-30 mg/L of immunoreactive peptides was shown to be correctly processed and to possess biological activity as measured in vitro and in vivo. Additional peptides purified from broth appear to result from proteolytic degradation of the original translation product. Analysis of the amino acid compositions and sequences of these peptides suggests that processing enzymes may be responsible for some of the degradation.

Solubilization of rat lung vasoactive intestinal peptide receptors in the active state. Characterization of the binding properties and comparison with membrane-bound receptors.

We demonstrate here that rat lung membrane vasoactive intestinal peptide (VIP) receptors can be extracted in the active state using digitonin. Sepharose 4B gel filtration chromatography was utilized to demonstrate the formation of specific binding complexes between 125I-VIP and solubilized receptors. A rapid soluble receptor assay was established to separate 125I-VIP-receptor complexes from free 125I-VIP, which entailed differential precipitation of the 125I-VIP-receptor complex with polyethylene glycol and bovine gamma-globulin. Using this assay, several detergents were tested for their suitability to extract active VIP receptors, and most favorable results were obtained with digitonin, as judged by specific binding of 125I-VIP to the solubilized receptors. Time course studies indicated that the binding of 125I-VIP to digitonin extract was more rapid than to rat lung membranes. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract, as in the membrane. The values for the dissociation constants (Kd) were 200 pM for Class I and 8 nM for Class II receptors while the values for binding capacity (Bmax) were 200 and 2300 fmol/mg for Class I and II sites, respectively. Although the binding parameters of the two classes were similar to those in the membrane, the pharmacological properties were different, as evidenced by the inability of rat growth hormone releasing factor, a potent VIP agonist in the membrane, to displace specifically bound 125I-VIP from solubilized receptors. The ability to solubilize active VIP receptors represents an important step toward purification of the functional protein.

Development of helix-based vasoactive intestinal peptide analogues: identification of residues required for receptor interaction.

Several VIP analogues have been designed on the basis of the hypothesis that the region from residue 6 to residue 28 forms a pi-helical structure when bound to membrane receptors. An empirical approach for the design and construction of analogues based upon distribution frequency and structural homology with several sequence-related peptides is presented. Five peptides were designed, synthesized, and analyzed. One analogue, model 5, containing the native hydrophobic and an altered hydrophilic surface, was an effective VIP agonist in both binding to rat lung membrane receptors (KD1 = 11 +/- 8 pM, KD2 = 6.4 +/- 0.2 nM; VIP KD1 = 21 +/- 13 pM, KD2 = 1.8 +/- 0.6 nM) and stimulation of amylase release from guinea pig pancreatic acini (ED50 = 90 pM; VIP ED50 = 27 pM). The four other analogues were considerably less potent than VIP, yet retained full intrinsic activity. Our results showed that the hydrophobic surface of this helical domain (residues 6-28) contains amino acids important for interaction with receptors, whereas amino acid residues on the hydrophilic surface do not seem to participate strongly in receptor binding or signal transduction. Furthermore, on the basis of high-affinity binding, the stimulation of amylase release in pancreatic acini appears to be coupled to the higher affinity receptors. These results suggest that an approach based on the construction of putative pi-helical structures can be applied to the design of biologically active analogues of VIP. Thus, we have identified several residues within the VIP sequence that are critical for receptor binding using this approach.

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.

Design and biological activity of analogs of growth hormone releasing factor with potential amphiphilic helical carboxyl termini.

A 29-amino acid analog of growth hormone releasing factor (GHRF) was designed in which the sequence of the first six amino acids at the amino terminus was maintained while the postulated amphiphilic helical structure in the remainder of the molecule was optimized. The amino acid sequence of the analog differed from that of the first 29 residues of human GHRF by 13 residues. The peptide was synthesized by the solid-phase procedure in amide and free acid forms, both of which were tested for biological activity. When assayed for the ability to stimulate growth hormone secretion in primary cultures of rat anterior pituitary cells, the amide analog was 1.57 times as potent as GHRF-(1-40)-OH, which was used as the standard for comparison, while the free acid form was 1/6th as potent in the same assay. The two forms of the analog were also tested for stimulation of cAMP formation; they exhibited relative potencies similar to those observed for growth hormone secretion. The high activity of the analog provides good evidence for the importance of an amphiphilic helical structure in the carboxyl-terminal portion of the GHRF molecule.

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.