The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the aminoterminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) are regulatory peptides of importance in a wide variety of cell physiologic processes including secretion, smooth muscle contraction, and modulation of neuron firing rate (for review, see refs. 1 and 2). In addition, bombesin-like peptides can function as growth factors in Swiss 3T3 murine embryonal fibroblasts (3) and have been implicated as autocrine growth factors in the pathogenesis of some human small cell lung carcinomas (4). The bioactivities associated with the mammalian bombesinlike peptides are mediated by high-afflinity binding to cell surface receptors present on many target cells (for review, see ref. 1). The properties of high-affinity bombesin/GRP receptors found in relatively high numbers (=100,000 receptors per cell) on Swiss 3T3 fibroblasts have been extensively studied (5), with subsequent purification of the protein to near homogeneity (6). Molecular cloning ofthe gene encoding the Swiss 3T3 bombesin/GRP receptor is the next step to understanding the diversity and function of mammalian bombesin-like peptides and their receptors in physiologic and pathologic processes. In this paper, we report the isolation and characterization of cDNA clones § encoding the Swiss 3T3 bombesin/GRP receptor, defining the structure of the receptor polypeptide. The clones obtained should provide a basis for further molecular analysis ofthe structure, function, and expression of receptors in Swiss 3T3 cells. In addition, these clones provide a means for a similar analysis of this receptor and other related bombesin peptide receptors expressed in the various cell types known to respond to this family of peptides. MATERIALS AND METHODSReceptor Protein Purification and Peptide Sequencing. The ...

Abstract-This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mouse peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersions and were subsequently exposed to an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. This treatment produced a rapid accumulation of cellular FC. The FC that accumulated due to ACAT inhibition was more readily available for efflux to 2-hydroxypropyl-␤-cyclodextrin (which removes cholesterol from the plasma membrane) than FC in untreated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment.

The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-1 (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34cdc2 kinase site on BTG1 (Ser-159). In yeast, the Ala-159 mutant did not interact with hCAF-1. In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34CDK2/cyclin E and p34CDK2/cyclin A, but not for p34CDK4/cyclin D1 or p34cdc2/cyclin B. Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-1 (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 [Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera. Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67% and 90% respectively. Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact.