The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.

A brain polypeptide termed diazepam-binding inhibitor (DBI) and thought to be chemically and functionally related to the endogenous effector of the benzodiazepine recognition site was purified to homogeneity. This peptide gives a single band of protein on NaDodSO4 and acidic urea gel electrophoresis. A single UV-absorbing peak was obtained by HPLC using three different columns and solvent systems. DBI has a molecular mass of 411,000 daltons. Carboxyl-terminus analysis shows that tyrosine is the only residue while the amino-terminus was blocked. Cyanogen bromide treatment of DBI yields three polypeptide fragments, and the sequences of two of them have been determined for a total of 45 amino acids. DBI is a competitive inhibitor [3H]imipramine binding tested at their respective Kd values. DBI injected intraventricularly at doses of 5-10 nmol completely reversed the anticonflict action of diazepam on unpunished drinking and, similar to the anxiety-inducing 1-carboline derivative FG 7142 (13-carboline-3-carboxylic acid methyl ester), facilitated the shockinduced suppression of drinking by lowering the threshold for this response.

Pure bovine brain-derived acidic fibroblast growth factor is a very potent mitogen for vascular endothelial cells in culture and, in the presence of heparin, induces blood vessel growth in vivo. Partial Fibroblast growth factor (FGF) was originally identified in both pituitary (2) and whole brain (3). The growth factor activity was partially purified based on its ability to stimulate DNA synthesis in the immortal BALB/c 3T3 fibroblast cell line (4). Two mitogens, one acidic (5) and one basic (6), were subsequently identified in the partially purified brain FGF preparations. We previously reported the purification to apparent homogeneity and initial characterization of the approximately 17-kDa acidic FGF (aFGF) from bovine brain (7). A protein that may be similar, or identical, to the basic FGF found in brain has been purified from bovine pituitary (8, 9).With partially purified material, the presence of the acidic mitogenic activity for BALB/c 3T3 cells was observed to correlate with mitogenicity for vascular endothelial cells (6). We report here that aFGF is a potent mitogen for vascular endothelial cells in culture and induces the growth of blood vessels in vivo. Protein sequence data are also presented that uniquely identify this molecule and reveal homology with one type of interleukin 1 (IL-1). MATERIALS AND METHODSMitogenic Assays. Fetal bovine thoracic aortic endothelial cells (AG4762, National Institute of Aging Cell Repository, Institute for Medical Research, Camden, NJ) were assayed after 38 cumulative population doublings in vitro. The cells were plated in 6-well tissue culture dishes (Costar, Cam-bridge, MA) at 2 x 103 cells per cm2 in 1 ml of 20% heat-inactivated calf serum (GIBCO) in Dulbecco's modified Eagle's medium (DMEM; GIBCO) per well and changed to 1% serum 18 hr later. All media were supplemented with glutamine and penicillin/streptomycin as described (5). Either pure mitogen (7) diluted in 100 pl of 1 mg ofbovine serum albumin (Sigma) per ml of DMEM or serum samples were added to each well along with 1.6 puCi of [methyl-3H]thymidine (20.0 Ci/mmol, New England Nuclear; 1 Ci = 37 GBq) and 45 ,ug of unlabeled thymidine (Sigma) in 40 ,ul of DMEM. After a 48-hr incorporation period, the cells were washed and lysed, and 75% of the trichloroacetic acid-insoluble radiolabeled DNA was counted as described (5). The pure mitogen was found to be stable in the 7 mM trifluoroacetic acid/33% acetonitrile HPLC elution solvent (7) at -20°C under argon but lost substantial activity when lyophilized. Therefore, in all mitogenic and angiogenic assays, the pure mitogen was diluted from this solvent. Control assays showed that equivalent amounts of HPLC solvent components were innocuous.Mouse lung capillary endothelial cells (from T. Maciag, Revlon) were plated at 2.6 x 104 cells per cm2 in 24-well dishes (Costar) and grown to confluence in 0.5 ml of 10% charcoal-treated calf serum (HyClone, Logan, UT) in DMEM per well, lowered to 0.5% serum after 72 hr, and allowed to become quiescent over 48 hr. Eit...

Bovine brain-derived acidic fibroblast growth factor (aFGF) is a protein mitogen originally identified in partially purified preparations of whole brain. The protein was purified to homogeneity and shown to be a potent vascular endothelial cell mitogen in culture and angiogenic substance in vivo. The homology of aFGF to human interleukin-1 beta was inferred from partial sequence data. The complete amino acid sequence of aFGF has now been determined and observed to be similar to both basic FGF and interleukin-1's. A neuropeptide-like sequence, flanked by basic dipeptides, was observed within the aFGF sequence.