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.

We have isolated and sequenced a cDNA encoding the human 132-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 P2-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 P2-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.Many hormones, neurotransmitters, and drugs influence cellular metabolic activities by stimulating the adenylate cyclase system, leading to the generation of the second messenger cAMP and activation of the cAMP-dependent protein kinase. The molecular components of this plasma membrane signaling system include specific receptors that bind ligands, the catalyst that converts ATP to cAMP, and guanine nucleotide regulatory or G proteins that functionally couple the receptors to the enzyme (1). The latter two components of the system have been purified and genes encoding several members of the "G protein family" have been cloned.Of the receptors that are coupled to adenylate cyclase the only one that has been characterized in any detail is the p-adrenergic receptor (,8AR). Two pharmacologically and physiologically distinct subtypes of this receptor, termed ,31AR and f32AR, are both membrane glycoproteins of Mr 64,000 (2). Very recently, we reported cloning of cDNA and the gene for the hamster f32AR (3). The deduced protein sequence indicated a protein of 418 amino acids, with seven clusters of hydrophobic amino acids likely representing membrane-spanning regions. This topology resembles that of the visual "light receptor" rhodopsin, which also possesses seven membrane-spanning domains (4-6).We now report the cloning and complete nucleotide sequence of the cDNA for the human ,82AR. While the receptor is highly similar to its hamster counterpart (87% of the amino acid residues are identical), significant regional differences in the extent of identity are noted. METHODScDNA Library Screening. The human placenta cDNA library was kindly provided by Evan Sadler (Washington University School of Medicine). The cDNA was prepared from term placenta poly(A)+ RNA and cloned in phage Xgtll. The library contains 5 x 106 independent recombinants. The A431 Xgtll library was prepared from poly(A)+ RNA from actively dividing A431 cells by methods previously described (3). It contains 1 x 106 independent reco...

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 vine 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 =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.The 3T3-L1 cell line, isolated by Green and Kehinde (1, 2), represents a useful model system for investigating the mechanisms of cell differentiation. Under appropriate conditions, 3T3-L1 preadipocytes, which initially resemble fibroblasts, differentiate into adipocytes in cell culture (1-3). The differentiation process is accompanied by a dramatic increase in the activities of the enzymes associated with de novo lipogenesis (3-8) and triglyceride mobilization (9), as well as an increased sensitivity to lipogenic and lipolytic hormones (10-12). The cells accumulate triglyceride (1-3) and acquire the morphological characteristics of adipocytes (13) isolated from normal adipose tissue. There is good evidence that, for some of the lipogenic enzymes (e.g., fatty acid synthetase), the increases in activity during differentiation arise from increased rates of enzyme synthesis (6,14,15 MATERIALS AND METHODS Cell Culturing. 3T3-L1 preadipocytes were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum until differentiation was initiated according to the procedure of Reed and Lane (11). In this protocol, confluent cell monolayers were incubated for 48 hr in medium containing 10% fetal bovine serum/methylisobutylxanthine (115 ug/ml)/insulin (10 gg/ml)/dexamethasone (390 ng/ml). After 48 hr, the cells were washed free of methylisobutylxanthinie and dexamethasone and maintained in medium containing 10% fetal bovine serum and insulin at 10 ,ug/ml, at which time the cells express the adipocyte phenotype within 48 to 72 hr.Nucleic Acid Isolation and in Vitro Translations. Total cellular RNA was isolated by the procedures described by Chirgwin et al. (18) except that chloroform/butanol (4:1, vol/ vol) was used for deproteinization. The RNA was separated from any traces of DNA or protein by pelleting through CsCl (19). Poly(A)+-containing RNA was prepared as described by Aviv and Leder (20 5468The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.