Transmembrane topology of the mammalian KDEL receptor.

The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm.     

Distinct molecular mechanisms account for the specificity of two different T-cell receptors

Analysis of the thermodynamics of the interactions between the D3 T-cell receptor (TCR) and its natural ligand, an HIV peptide bound to a HLA-A0201 (HLA-A2) major histocompatibility complex (MHC) protein, shows both similarities and striking differences when compared with the 2B4 TCR binding to its peptide-MHC ligand. The equilibrium thermodynamic parameters of both reactions are consistent with a conformational adjustment at the binding interface during the formation of specific TCR-peptide-MHC complexes. However, osmolytic reagents that dehydrate protein surfaces have profoundly different effects on the strength of the two reactions, indicating that water molecules make very different contributions-enhancing the binding of D3 TCR but weakening the binding of 2B4 TCR. The use of these different mechanisms by TCRs to recognize ligands might be an important means augmenting their inherent cross-reactivity.     

A possible molecular mechanism for mammalian aging

The following model of aging is proposed: 1) defective proteins with anomalous primary structures are synthesized sometimes; 2) these defective proteins are precipitated in cells and intercellular spaces; 3) the precipitated proteins block them up under the influence of radicals; 4) a decrease of cell functional ability below some level results in the destruction of the organism regulation function. A formula is concluded connecting the life span (Tlife) with DNA-repair velocity (Vrep) and time of protein exchange (Tex): Tlife = (1/3).K.(Vper/Vdum).(Tfix + Tex), where K-admitted share of fixated proteins (fixated/native), Vdam-damage velocity, Tfix-fixation time of defective proteins. This analytical dependence was probed on literature data for man, elephant, cow, rabbit, guinea pig, golden hamster, rat, mouse and shrew. Tfix is shown to equal 5 divided by 10 days. A good agreement between the theoretical dependence Tlife(Tex) and literature data was obtained with the exception of the data for man.     

The cyclic nucleotide specificity of three cAMP receptors in Dictyostelium.

cAMP receptors mediate signal transduction pathways during development in Dictyostelium. A cAMP receptor (cAR1) has been cloned and sequenced (Klein, P., Sun, T. J., Saxe, C. L., Kimmel, A. R., Johnson, R. L., and Devreotes, P. N. (1988) Science 241, 1467-1472) and recently several other cAR genes have been identified (Saxe, C. L., Johnson, R., Devreotes, P. N., and Kimmel, A. R. (1991a) Dev. Genet. 12, 6-13; Saxe, C. L., Johnson, R. L., Devreotes, P. N., and Kimmel, A. R. (1991b) Genes Dev. 5, 1-8). We have expressed three receptor subtypes, cAR1, cAR2, and cAR3, in growing cells and have investigated their affinity and pharmacological specificity in a series of [3H]cAMP binding studies. In phosphate buffer, there were two affinity states of about 30 and 300 nM for cAR1 and 20 and 500 nM for cAR3 but no detectable affinity for cAR2. In the presence of 3 M ammonium sulfate, there was one affinity state of 4 nM for cAR1 and 11 nM for cAR2 and two affinity states of approximately 4 and 200 nM for cAR3. The relative affinities of 14 cyclic nucleotide derivatives were tested for each cAR in ammonium sulfate. These studies suggest a model (Van Haastert, P. J. M., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) in which cAMP binds to all three receptor subtypes by maintaining hydrogen bond interactions at the N6 and O3' positions. Interactions at the exocyclic oxygens of cAMP varied between the receptors; cAR2 and cAR3 lacked a stereoselective interaction at the axial oxygen which was present in cAR1. The cleft, which binds the adenine ring of cAMP, was hydrophobic in cAR1 and cAR3 but relatively polar in cAR2. The analog specificity of cAR1 and cAR3 in phosphate buffer was similar to that measured in ammonium sulfate though the derivatives' relative affinity to cAMP was reduced. We conclude that these cAMP receptor subtypes can be distinguished by distinct pharmacological properties which will allow selective activation of each cAR during development.     

A molecular marker for centriole maturation in the mammalian cell cycle

The centriole pair in animals shows duplication and structural maturation at specific cell cycle points. In G1, a cell has two centrioles. One of the centrioles is mature and was generated at least two cell cycles ago. The other centriole was produced in the previous cell cycle and is immature. Both centrioles then nucleate one procentriole each which subsequently elongate to full-length centrioles, usually in S or G2 phase. However, the point in the cell cycle at which maturation of the immature centriole occurs is open to question. Furthermore, the molecular events underlying this process are entirely unknown. Here, using monoclonal and polyclonal antibody approaches, we describe for the first time a molecular marker which localizes exclusively to one centriole of the centriolar pair and provides biochemical evidence that the two centrioles are different. Moreover, this 96-kD protein, which we name Cenexin (derived from the Latin, senex for "old man," and Cenexin for centriole) defines very precisely the mature centriole of a pair and is acquired by the immature centriole at the G2/M transition in prophase. Thus the acquisition of Cenexin marks the functional maturation of the centriole and may indicate a change in centriolar potential such as its ability to act as a basal body for axoneme development or as a congregating site for microtubule-organizing material.     

A novel family of mammalian taste receptors

In mammals, taste perception is a major mode of sensory input. We have identified a novel family of 40-80 human and rodent G protein-coupled receptors expressed in subsets of taste receptor cells of the tongue and palate epithelia. These candidate taste receptors (T2Rs) are organized in the genome in clusters and are genetically linked to loci that influence bitter perception in mice and humans. Notably, a single taste receptor cell expresses a large repertoire of T2Rs, suggesting that each cell may be capable of recognizing multiple tastants. T2Rs are exclusively expressed in taste receptor cells that contain the G protein alpha subunit gustducin, implying that they function as gustducin-linked receptors. In the accompanying paper, we demonstrate that T2Rs couple to gustducin in vitro, and respond to bitter tastants in a functional expression assay.     

Characterization of mammalian receptors for lactoferrin.

Lactoferrin (Lf) has been suggested to have several physiological functions. Specific binding of Lf, indicating the presence of Lf receptors (LfRs), has been observed in various types of mammalian cells such as lymphocytes, hepatocytes, and enterocytes. These LfRs are considered to function as a mediator for some of the functions of Lf. We here review current knowledge of mammalian LfRs characterized in different tissues. We also briefly present evidence for the existence of an LfR provided by our cloning of a human intestinal LfR (HLfR). The entire coding region of the HLfR was cloned by polymerase chain reaction (PCR), and a recombinant HLfR (rHLfR) was expressed in a baculovirus system. The rHLfR was purified by immobilized human Lf (HLf) affinity chromatography, indicating that the rHLfR retained the capacity to bind HLf. The gene was expressed at high levels in fetal small intestine and in adult heart but at lower levels in Caco-2 cells. In summary, we demonstrate the presence of a unique receptor-mediated mechanism for Lf, functioning in the small intestine of the newborn infant and possibly in other tissues of human adults.     

Estrogen receptors and androgen receptors in the mammalian liver

An estrogen receptor and an androgen receptor are present in the mammalian liver. In the liver of the rat, the estrogen receptor concentration increases markedly at puberty and this change correlates with enhanced estrogen stimulation of plasma renin substrate synthesis. High doses of estrogen are required for nuclear binding in liver when compared to doses for the uterus. The high dose requirement appears to be predominantly due to extensive metabolism in the hepatocyte of the estrogen to inactive derivatives. Furthermore, estradiol is much weaker than ethinyl estradiol for promoting nuclear binding in the liver. This is due to extremely rapid and extensive metabolism of estradiol. In human liver the concentration of estrogen receptor is low. An androgen receptor is present in high concentration in rabbit liver and is located predominantly in the nucleus after androgen administration. High concentrations of a putative androgen receptor are also present in human liver cytosol. Preliminary studies indicate that synthetic progestins can attach to the human liver androgen receptor. To date, a progesterone receptor has not been found in the mammalian liver. Thus, it appears that extensive steroid metabolism in liver preferentially diminishes sex steroid interaction with liver receptors and that androgen receptors may mediate progestin effects in liver. These observations provide a scientific basis for improved safety of oral contraceptives. Lowering the estrogen and progestin doses in oral contraceptives will decrease the major side-effects, which are liver mediated, and still maintain the desired effects at the hypothalamic-pituitary axis and uterus. Furthermore, it is likely that by selecting which estrogen, progestin or androgen is administered as well as by utilizing a parenteral route of administration that sex steroid effects on the liver could be minimized.     

Agonist derived molecular probes for A2 adenosine receptors

The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5'-N- ethylcarboxamidoadenos ine (CGS21680) was recently reported to be selective for the A2 adenosine receptor subtype, which mediates its hypotensive action. To investigate structure/activity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A2 adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A2 receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2 receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology.     

A molecular dissection of the glycoprotein hormone receptors

In glycoprotein hormone receptors, a subfamily of rhodopsin-like G protein-coupled receptors, the recognition and activation steps are carried out by separate domains of the proteins. Specificity of recognition of the hormones thyrotropin (TSH), lutropin (LH), human chorionic gonadotropin (hCG) and follitropin (FSH) involves leucine-rich repeats (LRRs) present in an N-terminal ectodomain, and can be associated with a limited number of residues at key positions of the LRRs. The mechanism by which binding of the hormones results in activation is proposed to involve switching of the ectodomain from a tethered inverse agonist to a full agonist of the serpentine, rhodopsin-like region of the receptor. Unexpectedly, the picture is complicated by the observation that promiscuous activation of one of the receptors (FSHr) by hCG or TSH can result from activating mutations affecting the serpentine region of the receptors.     

Nicotine receptors in the mammalian brain

Nicotine is a drug of abuse that presumably exerts its psychoactive effect through its interactions with nicotine binding sites in the central nervous system. Among its potential sites of action are the neuronal nicotinic acetylcholine receptors and the neuronal alpha-bungarotoxin binding sites. In this review we focus on the neuronal nicotinic acetylcholine receptors, their diversity, distribution, and functions as nicotine receptors or as mediators of synaptic transmission in the mammalian brain. We find that the complexity characteristic of the gene family encoding the subunits of these receptors is reflected both in the pattern of expression of the genes and in the pharmacological diversity of the expressed receptors.     

A molecular level prototype for mechanoelectrical transducer in mammalian hair cells

The mechanoelectrical transducer (MET) is a crucial component of mammalian auditory system. The gating mechanism of the MET channel remains a puzzling issue, though there are many speculations, due to the lack of essential molecular building blocks. To understand the working principle of mammalian MET, we propose a molecular level prototype which constitutes a charged blocker, a realistic ion channel and its surrounding membrane. To validate the proposed prototype, we make use of a well-established ion channel theory, the Poisson–Nernst–Planck equations, for three-dimensional (3D) numerical simulations. A wide variety of model parameters, including bulk ion concentration, applied external voltage, blocker charge and blocker displacement, are explored to understand the basic function of the proposed MET prototype. We show that our prototype prediction of channel open probability in response to blocker relative displacement is in remarkable accordance with experimental observation of rat cochlea outer hair cells. Our results appear to suggest that tip links which connect hair bundles gate MET channels.     

Origin of the genetic code and specificity of tRNA aminoacylation. A testable model

Conclusion The specificity of tRNA aminoacylation as well as the origin of the genetic code are far from being understood at the molecular and evolutionnary level. The tRNA-tRNA interaction model could provide a missing link for resolving both problems. The model suggests a direct chemical interaction between the nucleotides in the anticodon, and the amino acid (adenylate) to be transferred to the 3-terminal adenosine, within the catalytic center (23). The experimental data reviewed here indicate that in many, but not all, systems the anticodon does play a major role during the aminoacylation and that the simultaneous binding of two tRNA molecules for aminoacylation (of only one of them) does not contradict enzymatic and crystallographic data (24).     

Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.