M A Bolanowski

We have isolated and sequenced a cDNA encoding the human beta 2-adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster beta 2-adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. We have localized the gene for the beta 2-adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor.

Angiostatin, a cleavage product of plasminogen, has been shown to inhibit endothelial cell proliferation and metastatic tumor cell growth. Recently, the production of angiostatin has been correlated with tumor-associated macrophage production of elastolytic metalloproteinases in a murine model of Lewis lung cell carcinoma. In this report we demonstrate that purified murine and human matrix metalloproteinases generate biologically functional angiostatin from plasminogen. Macrophage elastase (MMP-12 or MME) proved to be the most efficient angiostatin-producing MMP. MME was followed by gelatinases and then the stomelysins in catalytic efficiency; interstitial collagenases had little capacity to generate angiostatin. Both recombinant angiostatin and angiostatin generated from recombinant MME-treated plasminogen inhibited human microvascular endothelial cell proliferation and differentiation in vitro. Finally, employing macrophages isolated from MME-deficient mice and their wild-type littermates, we demonstrate that MME is required for the generation of angiostatin that inhibits the proliferation of human microvascular endothelial cells.

To identify and characterize specific mRNAs that increase in abundance during differentiation of mouse 3T3-L1 preadipocytes, a cDNA library was constructed from poly(A)+RNA isolated from differentiated 3T3-L1 adipocytes. Mixed probe isotope ratio selection and RNA blot analyses have identified several unique cDNA clones that represent mRNA species expressed either exclusively or at dramatically increased levels in differentiated cells. Further characterization of one such clone (pAL422) revealed that the corresponding mRNA, detectable only after differentiation, is approximately the same length (600 +/- 150 bases) as the cDNA insert (672 bases). The complete nucleotide sequence of the cDNA insert in pAL422 revealed a single long open reading frame that encodes a 132 amino acid polypeptide (the 422 protein) of 14.6 kDa. These and other results suggest that this cDNA may represent a nearly full-length copy of the mRNA. Computer-assisted analyses showed that the 422 protein shares 69% and 64% homology with myelin P2 proteins from rabbit and bovine peripheral nerves, respectively, as well as 23% and 30% homology with fatty-acid binding proteins from rat liver and intestine, respectively. Moreover, the mRNA hybrid selected by pAL422 DNA directs the in vitro translation of an approximately equal to 13 kDa polypeptide, and this protein is specifically immunoprecipitated by antiserum against bovine myelin P2. These observations strongly suggest that the 422 protein is a structural, and possibly functional, analog of myelin P2.

Differentiation of 3T3-L1 preadipocytes in culture is accompanied by alterations in the abundance of several mRNAs and by the appearance of many new adipocyte-specific mRNAs. To investigate the processes responsible for these alterations, the kinetics of accumulation of several specific mRNAs were compared with their respective rates of nuclear runoff transcription. The mRNAs for fructose-1,6-bisphosphate aldolase and an unidentified 4800-base mRNA increase in abundance only moderately (2-4-fold) during differentiation. Runoff transcription by nuclei isolated from 3T3-L1 cells during the course of differentiation revealed very little or no change in the rates of transcription of these mRNAs. Similar results were obtained for the beta, alpha-actin and beta-tubulin mRNAs where no difference in nuclear runoff transcription rates were observed even though a 2-fold decrease in the steady-state levels of these mRNAs accompanies differentiation. In contrast, the steady-state levels of mRNAs for 3T3-L1 P2 protein, an adipocyte homologue of myelin P2 protein, and an unidentified 5000-base mRNA increased dramatically (greater than 20-fold) during adipose conversion. These large increases in abundance were correlated with marked rises (greater than 10-fold) in nuclear runoff transcription rates for these mRNAs during differentiation of 3T3-L1 preadipocytes. No change in runoff transcription activity for these mRNAs was detected by nuclei from control nondifferentiating 3T3-C2 cells. These results strongly suggest that an increased rate of specific transcription is primarily responsible for the accumulation of these mRNAs during preadipocyte differentiation.