Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coHl and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.

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.

The crystal structure of the protease of the human immunodeficiency virus type (HIV-1), which releases structural proteins and enzymes from viral polyprotein products, has been determined to 3 A resolution. Large regions of the protease dimer, including the active site, have structural homology to the family of microbial aspartyl proteases. The structure suggests a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation.

The 21-kilodalton protein (p21) encoded by normal cellular Harvey-ras has been expressed in Escherichia coli as a fusion protein by using the pUC8 vector and has been purified to >95% homogeneity by ion-exchange chromatography and gel filtration. The purified protein molecules possess intrinsic GTPase activity on the basis of the following criteria: (i) elution of the GTPase activity with p21 GDP-binding activity in two different chromatography systems, (it) parallel thermal inactivation of GTPase activity and p21 GTP-binding activity, and (iiM) immunoprecipitation of the GTPase activity with monoclonal antibodies to p21. At 37°C, the rate of GTP hydrolysis by the purified normal p21 assayed in solution was 5.3-6.6 mmol/min per mol of p21. The rate of GTP hydrolysis by a form of p21 [Val'2] encoded by a human oncogene was significantly lower (1.4-1.9 mmol/min per mol of p21). The presence of a threonine phosphate acceptor site at residue 59 also decreased p21 GTPase activity. For regulatory proteins that use GTP as part of their biochemical mechanism, the hydrolysis of GTP to GDP reverses the biological activity of the respective proteins. The observation that oncogenic forms of p21 lose GTPase activity suggests that GTP hydrolysis may be a biochemical event that inactivates the growth-promoting effects of a p21GTP complex.The ras oncogenes constitute a multigene family that transform cells through the action of a 21-kilodalton protein termed p21 (1, 2). p21 can be detected in normal mammalian cells (3), and ras-related genes and proteins are found in Drosophila and yeast (4, 5). These observations provide evidence that ras-encoded proteins may have essential and evolutionarily conserved normal cellular functions. Transformation of some mammalian cells can occur if p21 is expressed at an abnormally high level (6, 7) or if mutations of the ras gene alter the p21 primary structure (2,(8)(9)(10). A particularly potent mutation is one that changes a glycine at residue 12 of normal p21 to a valine in the oncogenic form of p21 (8).p21 interacts with the plasma membrane and lipid molecules (11, 12), binds guanine nucleotides specifically (13,14), and possesses an autophosphorylating activity if a threonine phosphate acceptor replaces the alanine at cellular p21 residue 59 (14-16). Previously, we demonstrated that p21 autophosphorylating activity distinguished the glycine form of p21 from the valine form in vitro and in intact NIH 3T3 mouse fibroblast cells (17). Since cellular p21 molecules with an alanine at residue 59 do not autophosphorylate (16), we speculated (17) that autophosphorylating activity may be a marker for some other p21 property such as GTPase activity. GTPase activity is a common property of other guanine nucleotide-binding proteins, including elongation factor (EF)-G (18, 19), 20), tubulin (21), and the membranebound G components of the adenylate cyclase (22) MATERIALS AND METHODSDNA Biochemistry. Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs. The large (Kleno...

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.

The functional significance of conserved polar amino acids within the putative transmembrane region of the fi-adrenergic receptor (PiAR) was examined by oligonucleotide-directed mutagenesis of the hamster gene encoding PAR and expression of the mutant genes in COS-7 cells. Although a substitution of aspartate at position 113 with an asparagine residue did not affect expression or processing of the protein, the resulting mutant BAR did not show detectable binding toward the antagonist iodocyanopindolol. Replacement of the aspartate and asparagine residues at positions 79 and 318, respectively, had no effect on the affinity of the receptor toward antagonists but reduced the affinity of the receptor toward agonists by 1 order of magnitude. Furthermore, we observed that substitution of the proline at position 323 with a serine residue resulted in improper or incomplete processing of the .BAR, presumably reflecting a role for this residue in the folding of the receptor. Together with our previous results from deletion mutagenesis studies, these observations indicate that the ligand binding site involves the transmembrane region of the BAR. The recent cloning of the genes encoding the hamster (2) and human 32AR (3), the avian PAR (4), and the porcine Ml muscarinic cholinergic receptor (MAR) (5) and the deduction of the primary sequences of these proteins suggest a structural basis for the mechanistic similarities among the Gprotein-linked receptors. These hormone receptors share sequence homology with each other and with the visual opsins, which transduce their signals through the activation of the G-protein transducin (6). Most of the conservation in sequence among these proteins is found within seven hydrophobic domains of approximately 20-25 residues in length. Based on the model proposed for the opsins (7), these hydrophobic regions of the receptors, which are linked by more divergent hydrophilic regions of various lengths, would alternately traverse the membrane with the N terminus of the PAR exposed externally and the C terminus exposed intracellularly (2). 4384The 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.

Receptors that mediate their actions by stimulating guanine nucleotide binding regulatory proteins (G proteins) share structural as well as functional similarities. The structural motif characteristic of receptors of this class includes seven hydrophobic putative transmembrane domains linked by hydrophilic loops. Genetic analysis of the beta-adrenergic receptor (beta AR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein. On the basis of these studies, a model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Other interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the beta AR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphiphilic alpha helix. This region of the beta AR is also critical for sequestration, which accompanies agonist-mediated desensitization, to occur. Structural similarities among G protein-linked receptors suggest that the information gained from the genetic analysis of the beta AR should help define functionally important regions of other receptors of this class.

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...