Irving S. Sigal

Structural basis of beta-adrenergic receptor function.

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 β‐adrenergic receptor (βAR) 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 βAR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphi‐philic α helix. This region of the βAR 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 βAR should help define functionally important regions of other receptors of this class.— Strader, C. D.; Sigal, I. S.; Dixon, R. A. F. Structural basis of β‐adrenergic receptor function. FASEB J. 3: 1825‐1832; 1989.

Cloning of bovine GAP and its interaction with oncogenic ras p21

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)1–3. 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 (ref. 4). GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21 (refs 3, 5 and 6). We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity7. 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 cerevisiae8–10 and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases26,27.

Structural features required for ligand binding to the beta-adrenergic receptor.

On the basis of the homology between the amino acid sequences of the beta‐adrenergic receptor (beta AR) and the opsin proteins we have proposed that the ligand binding domain lies within the seven transmembrane hydrophobic regions of the protein, which are connected by hydrophilic regions alternatively exposed extracellularly and intracellularly. We have systematically examined the importance of each of these regions by making a sequential series of deletions in the gene for the hamster beta AR which encompass most of the protein coding region. The ability of the corresponding mutant receptors to be expressed, localized to the cell membrane, and bind beta‐adrenergic ligands has been analyzed, using transient expression in COS‐7 cells. The hydrophobic regions and the hydrophilic segments immediately adjacent to the membrane cannot be removed without affecting the processing and membrane localization of the beta AR. However, most of the hydrophilic regions appear to be dispensable for ligand binding. In addition, we observed that substitution of the conserved cysteine residues at positions 106 and 184 dramatically altered the ligand binding characteristics of the beta AR, suggesting the occurrence of a disulfide bond between these two residues in the native protein. These data are discussed in terms of the tertiary structure of the beta AR.

Ligand binding to the β-adrenergic receptor involves its rhodopsin-like core

Recently the genes for several hormone receptors that interact with guanine nucleotide binding proteins (G proteins) have been cloned, including the hamster β2-adrenergic receptor (β2AR)1, a human β2AR2, the turkey erythrocyte β2AR3 and the porcine mus-carinic acetylcholine receptor (MAR)4. All these receptors share some amino-acid homology with rhodopsin, particularly in 7 hydro-phobic stretches of residues that are believed to represent trans-membrane helices5. To determine whether differences in ligand specificity result from the divergence in the sequences of the hydrophilic regions of these receptors, we have expressed in mammalian cells genes for the wild-type hamster and human βAR proteins, and a series of deletion mutant genes of the hamster β2AR. The pharmacology of the expressed receptors indicates that most of the hydrophilic residues are not directly involved in the binding of agonists or antagonists to the receptor. In addition, we have identified a mutant receptor that has high agonist affinity but does not couple to adenylate cyclase.

HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins.

The mature proteins of retroviruses originate as a result of proteolytic cleavages of polyprotein precursors. Retroviruses encode proteases responsible for several of these processing events, making them potential antiviral drug targets. A 99-amino acid HIV-1 protease, produced by chemical synthesis or by expression in bacteria, is shown here to hydrolyze peptides corresponding to all of the known cleavage sites in the HIV-1 gag and pol polyproteins. It does not hydrolyze peptides corresponding to an env cleavage site or a distantly related retroviral gag cleavage site.

The carboxyl terminus of the hamster β-adrenergic receptor expressed in mouse L cells is not required for receptor sequestration

The structural basis for agonist-mediated sequestration and desensitization of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster beta AR gene and expression of the mutant genes in mouse L cells. Treatment of these cells with the agonist isoproterenol corresponded to a desensitization of beta AR activity. A mutant receptor that bound agonist but did not couple to adenylate cyclase showed a dramatically reduced sequestration response to agonist stimulation. In contrast, beta AR mutants in which the C-terminus was truncated and/or in which two regions that have been proposed as phosphorylation substrates for cAMP-dependent protein kinase were removed showed normal sequestration responses. These results demonstrate that agonist-mediated sequestration of the beta AR can occur in the absence of the C-terminus of the protein and reveal a strong correlation between effective coupling to Gs and sequestration.

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C‐terminal 343 amino acids of GAP (residues 702‐1044) were observed to bind Ha ras p21‐GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M‐1 s‐1 at 24 degrees C) as GAP purified from bovine brain or full‐length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986‐1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C‐II, the crk oncogene product, and the non‐receptor tyrosine kinase oncogene products. This homologous region is located in the N‐terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.

Biochemical properties of normal and oncogenic ras p21

Abstract The ras proteins become highly oncogenic as a result of single amino acid mutations that inhibit GTP hydrolytic activity. Oncogenic p21 may be part of a growth-promoting complex that has lost its regulatory capabilities.

Affinity purification of the HIV-1 protease

An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme. A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi[CH2N]-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin. The HIV-1 protease was expressed in E. coli and the supernatant from lysed cells was passed through the affinity resin. Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl. Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography. Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column.

Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.