One of the assumptions of the mobile receptor hypothesis as it relates to G protein-coupled receptors is that the stoichiometry of receptor, G protein, and effector is 1:1:1 (Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125-132). Many studies on the cooperativity of agonist binding are incompatible with this notion and have suggested that both G proteins and their associated receptors can be oligomeric. However, a clear physical demonstration that G protein-coupled receptors can indeed interact as dimers and that such interactions may have functional consequences was lacking. Here, using differential epitope tagging we demonstrate that ␤ 2 -adrenergic receptors do form SDSresistant homodimers and that transmembrane domain VI of the receptor may represent part of an interface for receptor dimerization. The functional importance of dimerization is supported by the observation that a peptide derived from this domain that inhibits dimerization also inhibits ␤-adrenergic agonist-promoted stimulation of adenylyl cyclase activity. Moreover, agonist stimulation was found to stabilize the dimeric state of the receptor, while inverse agonists favored the monomeric species, which suggests that interconversion between monomeric and dimeric forms may be important for biological activity.

Domains rich in sphingolipids and cholesterol, or rafts, may organize signal transduction complexes at the plasma membrane. Raft lipids are believed to exist in a state similar to the liquid-ordered phase. It has been proposed that proteins with a high affinity for an ordered lipid environment will preferentially partition into rafts (Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G., and Brown, D. A. (1999) J. Biol. Chem. 274, 3910 -3917). We investigated the possibility that lipid-lipid interactions between lipid-modified proteins and raft lipids mediate targeting of proteins to these domains. G protein monomers or trimers were reconstituted in liposomes, engineered to mimic raft domains. Assay for partitioning of G proteins into rafts was based on Triton X-100 insolubility. Myristoylation and palmitoylation of G␣ i were necessary and sufficient for association with liposomes and partitioning into rafts. Strikingly, the amount of fatty-acylated G␣ i in rafts was significantly reduced when myristoylated G␣ i was thioacylated with cis-unsaturated fatty acids instead of saturated fatty acids such as palmitate. Prenylated ␤␥ subunits were excluded from rafts, whether reconstituted alone or with fatty-acylated ␣ subunits. These results suggest that the structural difference between lipids that modify proteins is one basis for the selectivity of protein targeting to rafts.Our view of the lateral organization of plasma membrane constituents has evolved in recent years from the conventional picture of membrane proteins diffusing freely in a sea of lipid (1). A large body of evidence from studies using cell biological and biophysical approaches suggests that there is selective confinement of lipids and proteins in discrete regions of the membrane (2-4). These domains, named lipid rafts, are rich in sphingolipids and cholesterol, and appear to be a ubiquitous feature of mammalian cells. Lipid rafts are likely to contribute to the structure and function of caveolae, plasma membrane invaginations that are implicated in a variety of cellular processes, including signal transduction, endocytosis, transcytosis, and cholesterol trafficking. It has been proposed that the spatial concentration of specific sets of proteins increases the efficiency and specificity of signal transduction by facilitating interactions between proteins and by preventing inappropriate cross-talk between pathways.Raft lipids have been proposed to exist in a separate phase from the rest of the bilayer, in a state similar to the liquidordered (l o )1 phase described in model membrane (5, 6). Acyl chains of lipids in the l o phase are tightly packed and highly ordered and extended, similar to those in the gel phase. Thus, lipid structural features (such as saturated acyl chains) that enhance formation of the gel phase can also enhance formation of the l o phase when these lipids are mixed with cholesterol. The presence of unusually long saturated acyl chains on sphingolipids promotes phase separation and formation of the l o phase in mixtures ...

Exposure of beta 2‐adrenergic receptors to agonists causes a rapid desensitization of the receptor‐stimulated adenylyl cyclase, associated with an increased phosphorylation of the receptor. Agonist‐promoted phosphorylation of the beta 2‐adrenergic receptor (beta 2AR) by protein kinase A and the beta‐adrenergic receptor kinase (beta ARK) is believed to promote a functional uncoupling of the receptor from the guanyl nucleotide regulatory protein Gs. More recently, palmitoylation of Cys341 of the receptor has also been proposed to play an important role in the coupling of the beta 2‐adrenergic receptor to Gs. Here we report that substitution of the palmitoylated cysteine by a glycine (Gly341 beta 2 AR) using site directed mutagenesis leads to a receptor being highly phosphorylated and largely uncoupled from Gs. In Chinese hamster fibroblasts (CHW), stably transfected with the human receptor cDNAs, the basal phosphorylation level of Gly341 beta 2AR was found to be approximately 4 times that of the wild type receptor. This elevated phosphorylation level was accompanied by a depressed ability of the receptor to stimulate the adenylyl cyclase and to form a guanyl nucleotide‐sensitive high affinity state for agonists. Moreover, exposure of this unpalmitoylated receptor to an agonist did not promote any further phosphorylation or uncoupling. A modest desensitization of the receptor‐stimulated adenylyl cyclase response was observed but resulted from the agonist‐induced sequestration of the unpalmitoylated receptor and could be blocked by concanavalin A. This contrasts with the agonist‐promoted phosphorylation and uncoupling of the wild type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Acanthocheilonema viteae is a parasitic nematode of rodents. We identified the chitinase of A. viteae infective stage larvae (L3) as the main target of the humoral immune response of jirds, which were protected against challenge infection after vaccination with irradiation attenuated L3. The cDNA of the L3 chitinase has been sequenced, and the deduced amino acid sequence shows significant homologies to chitinases of Brugia malayi microfilariae, insects, yeast, bacteria, and Streptomyces sp. The protein has been characterized by monoclonal antibodies and substrate activity gels. The chitinase of L3 may contribute to degrading the nematode cuticle during molting and thus represents a target of protective immune responses in a phase where the parasite is highly vulnerable. In addition, it has been shown that a similar enzyme exists in uterine microfilariae, which probably has a role in casting the egg shell.

The insulin-like growth factor (IGF) axis has been implicated in the progression of malignant disease and identified as a clinically important therapeutic target. Several IGF-1 receptor (IGF-1R) targeting drugs including humanized monoclonal antibodies have advanced to phase II/III clinical trials, but to date, have not progressed to clinical use, due, at least in part, to interference with insulin receptor signalling. We previously reported on the production of a soluble fusion protein consisting of the extracellular domain of human IGF-1R fused to the Fc portion of human IgG1 (first generation IGF-TRAP) that bound human IGF-1 and IGF-2 with a 3 log higher affinity than insulin. We showed that the IGF-TRAP had potent anti-cancer activity in several pre-clinical models of aggressive carcinomas. Here we report on the re-engineering of the IGF-TRAP with the aim of improving physicochemical properties and suitability for clinical applications. We show that cysteine-serine substitutions in the Fc hinge region of IGF-TRAP eliminated high-molecular-weight oligomerized species, while a further addition of a flexible linker, not only improved the pharmacokinetic profile, but also enhanced the therapeutic profile of the IGF-TRAP, as evaluated in an experimental colon carcinoma metastasis model. Dose-response profiles of the modified IGF-TRAPs correlated with their bio-availability profiles, as measured by the IGF kinase-receptor-activation (KIRA) assay, providing a novel, surrogate biomarker for drug efficacy. This study provides a compelling example of structure-based re-engineering of Fc-fusion-based biologics for better manufacturability that also significantly improved pharmacological parameters. It identifies the re-engineered IGF-TRAP as a potent anti-cancer therapeutic.