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Some strains of rats and mice express increased momentary fear or emotionality when exposed to a novel environment. Previous studies have found significantly fewer diazepam binding sites in the brains of Balb/cJ mice compared to C57BL and AKR/J mice and this has been suggested to contribute to the increased emotionally of the 'nervous' Balb strain. The influx of 36Cl into cerebral cortical microsacs was used to functionally assess the effects of GABA, diazepam and pentobarbital in the Balb mice compared to nonemotional animals (C57 and ICR mice). Slight differences in the ability of GABA to increase chloride influx were found among the three strains. Pentobarbital potentiation of GABA-mediated chloride flux was slightly higher in the ICR mice compared to Balb and C57. Diazepam potentiation of the effects of GABA, however, was significantly decreased in the Balb mice, strengthening the hypothesis that the benzodiazepine receptor is involved in mediating animal emotionality.

This study presents evidence for the conservation of Drosophila per gene homologs in mammalian DNA and for their expression in a number of tissues which are involved in various aspects of circadian timekeeping. Distinct 5 kb sequences, which hybridized to a non repetitive fragment of the Drosophila per gene under stringent conditions, were detected by Southern blotting. Sequences homologous to per gene of Drosophila were also amplified from rat and mouse brain cDNA libraries and from a mouse anterior hypothalamus and human hypothalamus libraries. Degenerate PCR primer design was based on conserved segments of the per protein. The per homologs were shown directly (by RT-PCR) to be expressed in hamster and mouse SCN, in hamster heart and in Aplysia and Bulla eyes.

We report the cloning of a human gene encoding the 5-hydroxytryptamine1B receptor. The receptor has the characteristics of a G-protein-linked receptor and is most homologous to the human 5-HT1D receptor. This human 5-HT receptor gene, most abundantly expressed in striatum, is localized on chromosome 6, at 6q13, and the gene encoding the 5-HT1D receptor is localized on chromosome 1. Radioligand studies indicate that the affinity of [3H]5-HT is 16 +/- 2 nM. Drug competition studies indicate that the receptor displays high affinity (i.e. less than 40 nM) for 5-HT, 5-carboxyamidotryptamine, methiothepin, and metergoline.

Using 32P-labelled oligonucleotides derived from the coding regions of dopamine D1, D2 and D3 receptor mRNAs we localized cells containing transcripts for these receptors in the human (hD1, hD2) and rat brain (rD1, rD2, rD3). Dopamine D1 receptor mRNA was detected at high levels in neurons of the caudate and putamen as well as in the nucleus accumbens in both human and rat brain. In the rat brain D1 receptor mRNA was also abundant in the olfactory tubercles and several thalamic nuclei. In both species D1 mRNA was absent from the neurons of the substantia nigra and the ventral tegmental area as well as from the globus pallidus medialis in humans and entopeduncular nucleus in rats. In contrast, dopamine D2 receptor mRNA was found in dopaminergic neurons of the substantia nigra pars compacta and of the ventral tegmental area. In addition high levels of D2 mRNA were detected in neurons of the caudate, putamen and accumbens nuclei, the olfactory tubercle and the anterior lobe of pituitary gland. In the rat the highest level of hybridization was found in the intermediate lobe of the pituitary gland. In the rat brain dopamine D3 mRNA was mainly detected in the Islands of Calleja and at lower levels in the anterior nucleus accumbens, the medial mammillary nucleus as well as in the bed nucleus of the stria terminalis. In general, a good agreement was found between the distribution of transcripts and binding sites labelled with the D1 antagonist SCH 23390 or with the D2 ligand SDZ 205-502. For D1 receptors, the main exceptions were the absence of mRNA in the globus pallidus and the substantia nigra despite the high densities of binding sites in these regions. For D2 receptors, regions where binding sites but not mRNA were detected included the olfactory bulb, neocortex, hippocampus and superior colliculus.

Molecular cloning studies have now identified five structurally homologous genes encoding the biosynthesis of the human dopamine receptors, DRD1, DRD2, DRD3, DRD4, and DRD5. Two of these dopamine receptors (DRD1 and DRD5) are encoded by intronless genes. To ascertain whether there are other intronless genes that share identity with the gene (DRD5) encoding the DRD5 receptor, we used a cloning method based on the polymerase chain reaction (PCR). Human genomic DNA was amplified by PCR with oligodeoxyribonucleotides (oligos) based on the DRD5 nucleotide (nt) sequence. Amplification of nt sequences between these oligos allowed the isolation of two independent intronless genes that share identity with DRD5. The full-length clones have also been isolated by screening human genomic libraries. The deduced amino acid sequences for these genes, PG-1 and PG-2, share 91% and 92% identity to DRD5, respectively. However, each of the genes contains differences in the coding regions that would render these genes incapable of encoding functional receptors. Thus, the human genome contains at least two DRD5 pseudogenes, consistent with in situ human chromosomal hybridization analysis which reveals the presence of two pseudogenes.

We have previously reported that the human genome contains the two pseudogenes psi DRD5-1, and psi DRD5-2, and that each share 94% homology when compared with the functional gene DRD5. There is only 2% difference at the nucleotide level between the two pseudogenes. We questioned whether these pseudogenes were transcribed, since transcription of either of these pseudogenes could result in false interpretation of in-situ hybridization and Northern blot analysis, using the DRD5 as a probe. We now report that we have detected transcription of one of the pseudogenes, psi DRD5-1, in several human brain areas, and this mRNA transcript is capable of producing a protein of 154 amino acids. Furthermore we report that PCR amplification of DRD5 or the pseudogenes in human tissue can result in the formation of chimer artifacts due to the co-amplification of three very similar genes.

Selectively outbred Dahl salt-sensitive (DS) and salt-resistant (DR) rats were compared with the inbred Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats developed from the original Brookhaven stocks by J.P. Rapp. The animals were evaluated for genotype at the renin locus. The inbred strains are uniformly homozygous for their respective alleles, s in SS/Jr and r in SR/Jr. DR rats were also uniformly homozygous for the r renin allele. In DS rats, however, three renin alleles were segregating. In addition to the s and r alleles, a third allele, designated the z allele, was found. The gene frequencies in DS rats were s = 0.690, r = 0.284, and z = 0.026. Continued use of DS and DR rats in most experimental work is inappropriate because of genetic heterogeneity in the DS stock.

Using 32P-labeled oligonucleotides derived from the coding region of human dopamine D1 receptor mRNA we have localized in the human and rat brain the cells containing the mRNAs coding for this receptor. Dopamine D1 receptor mRNA in human brain was found to be contained in the neurons of the caudate and putamen nuclei as well as in the nucleus accumbens, some cortical regions and some nuclei of the amygdala. In the rat brain, cells containing D1 receptor mRNA were enriched in caudate-putamen and accumbens nuclei, olfactory tubercle, islands of Calleja, some cortical areas and in several thalamic nuclei. Moreover, in both species, it was absent from the neurons of the substantia nigra both pars compacta and pars reticulata and ventral tegmental area as well as from the globus pallidus pars lateralis and medialis in human and globus pallidus and entopeduncular nucleus in rat. In general, a good agreement was found with the distribution of binding sites labeled with the D1 antagonist SCH 23390. The main exception was the absence of D1 receptor mRNA in globus pallidus and substantia nigra, regions where high densities of receptor sites are found. These data support the notion that sites in these two regions are localized to projections from striatal neurons and that dopaminergic neurons do not express this receptor.

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.

Physiological and pharmacological criteria have divided dopamine receptors into D1 and D2 subtypes, and genes encoding these subtypes have recently been cloned. Based on the sequences of the cloned receptors, we prepared oligodeoxynucleotide probes to map the cellular expression of the corresponding mRNAs in rat brain by in situ hybridization histochemistry. These mRNAs showed largely overlapping yet distinct patterns of expression. The highest levels of expression for both mRNAs were observed in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Within the caudate-putamen, 47 +/- 6% and 46 +/- 5% of the medium-sized neurons (10-15 microns) expressed the D1 and D2 mRNAs, respectively, and only the D2 mRNA was observed in the larger neurons (greater than 20 microns). The D1 and D2 mRNAs were expressed in most cortical regions, with the highest levels in the prefrontal and entorhinal cortices. Within neocortex, D1 mRNA was observed primarily in layer 6 and D2 mRNA in layers 4-5. Within the amygdala, D1 mRNA was observed in the intercalated nuclei, and D2 mRNA in the central nucleus. Within the hypothalamus, D1 mRNA was observed in the suprachiasmatic nucleus and D2 mRNA in many of the dopaminergic cell groups. Within the septum, globus pallidus, superior and inferior colliculi, mammillary bodies, and substantia nigra only D2 mRNA was detected. These data provide insight into the neuroanatomical basis of the differential effects of drugs that act on D1 or D2 receptors.

A rat renin allele (the S-allele) has been identified in Dahl rats which cosegregates with increases in blood pressure. Rats with a double dose of the allele--the salt-sensitive hypertensive rats--have low renin activity compared with the salt-resistant hypertensive rat that does not have this S-allele. Alcohol consumption in rats has also been shown to vary with renin activity, and the possible involvement of renin activity in the genetics of alcohol consumption was suggested by previous work showing that the alcohol-preferring P line of selected rats had low renin levels. In the present study we examined alcohol consumption in a group of inbred Dahl rats, which have a double dose of the S renin allele, and in a group of selected Dahl rats, which have only a single dose of this S-allele. After an initial acclimation period, these two groups were first given daily 1-hr access to ascending concentrations of alcohol (3%, 6%, 8% w/v) over a 34-day period followed by continuous access to alcohol for a further 10 days. Water and food were always available. Regardless of whether alcohol was rationed or continuously available, the rats with the double dose of the S-allele drank significantly more alcohol than the rats that had only a single dose of this allele. These findings suggest that genetically mediated alterations in the renin gene may exert a significant influence on alcohol consumption and may be a component in the etiology of alcoholism.

The integrity of coupling of the beta 2-adrenergic receptor (beta 2AR) to its guanine nucleotide-binding protein, Gs, and phosphorylation events on the receptor molecule have been proposed to be important determinants in the processes of receptor sequestration and down-regulation. However, little is known about the molecular mechanisms underlying these processes, and the regions of the receptor molecule that specifically subserve sequestration and down-regulation have yet to be delineated. To address these questions, we stably transfected eight mutant beta 2AR genes into Chinese hamster fibroblasts and evaluated the coupling, sequestration, and down-regulation properties of the mutated receptors. These mutant receptors have been previously demonstrated either to exhibit abnormal coupling to Gs or to lack functionally important phosphorylation sites for either the cAMP-dependent protein kinase or the agonist-dependent beta-adrenergic receptor kinase. All eight mutants exhibited receptor sequestration equivalent in extent to that of the beta 2AR, regardless of their coupling or phosphorylation status. However, four mutants that exhibited various degrees of impairment in coupling to Gs showed blunted receptor down-regulation patterns. Simultaneous treatment with isoproterenol and dibutyryl-cAMP did not improve the abilities of the mutant receptors to undergo down-regulation. These findings demonstrate that a variety of mutant beta 2AR with impaired coupling to Gs are, nevertheless, able to be sequestered normally. In contrast, agonist-induced down-regulation appears to require coupling of the beta 2AR to Gs but is largely independent of the generation of cAMP. Our results also suggest that molecular determinants of the beta 2AR involved in receptor sequestration are distinct from those participating in the down-regulation process.

Using the polymerase chain reaction to amplify genomic DNA from hair roots, we have examined the mitochondrial aldehyde dehydrogenase (ALDH2) genotypes of 28 individuals from the South American Mapuche Indians. We have determined that individuals from this population previously reported to lack (ALDH2) activity do not show the presence of the inactive (ALDH2(2] allele frequently found in Orientals.

An approach based on the polymerase chain reaction (PCR) was used to isolate additional members of the G-linked receptor family from a rat striatal lambda gtII cDNA library. Priming with one degenerate probe corresponding to highly conserved consensus sequences in the third transmembrane (TM) domain of 15 G-linked receptors and sequences in the phage vector resulted in one clone (G-13) encoding a dopamine D2 receptor variant with a 29 amino acid insert in the third cytoplasmic loop. In addition, the amino acid sequence encoded by clone G-36 contained conserved sequences characteristic of the G-linked class of receptors and displayed sequence homology in TM domains with the beta 2-adrenergic receptor (48%). Two conserved serine residues in TM5 postulated to be part of a ligand binding site in the adrenergic receptor, suggests that G-36 encodes a catecholaminergic receptor. Northern blot analysis confirmed the expression of G-36 in rat brain, but not in kidney, heart and lung. Several strong hybridizing bands to G-36 were obtained in both human and rat genomic DNA. The general PCR strategy employed here should prove to be extremely useful for the isolation of other members of the G-linked receptor family.

Activation of guanyl nucleotide regulatory proteins (G proteins) by hormones and neurotransmitters appears to require the formation of high affinity agonist-receptor-G protein ternary complexes. In the case of the beta 2-adrenergic receptor, multiple regions of the molecule have been implicated in coupling to the stimulatory G protein Gs. This finding raises the possibility that discrete regions of the receptor mediate ternary complex formation, whereas different loci may be involved in other aspects of G protein activation. To date, however, mutagenesis studies with the beta 2-adrenergic receptor have not clarified this question since mutant receptors with impaired abilities to activate Gs have generally possessed a diminished capacity to form the ternary complex as assessed in binding assays. We have expressed in a mammalian cell line a mutant beta 2-adrenergic receptor comprising a seven-amino acid deletion in the carboxyl-terminal region of its third cytoplasmic loop (D267-273), a region proposed to be critically involved in coupling to Gs. When tested with beta-adrenergic agonists, the maximal adenylyl cyclase response mediated by this mutant receptor was less than one-half of that seen with the wild-type receptor. Nevertheless, D267-273 exhibited high affinity agonist binding identical to that of the wild-type receptor. In addition, agonist-induced sequestration of the receptor, a property not mediated by Gs, was also normal. These findings indicate that the formation of high affinity agonist-receptor-Gs complexes is not sufficient to fully activate Gs. Instead, an additional stimulatory signal appears to be required from the receptor. Our data thereby suggest that the molecular determinants of the beta 2-adrenergic receptor involved in formation of the ternary complex are not identical to those that transmit the agonist-induced stimulatory signal to Gs.

The deduced protein sequences of the mammalian adrenergic receptors (ARs) suggest that these proteins have evolved by several ancient gene duplication events. To investigate in what species these events may have occurred DNA fragments encoding the family of adrenergic receptors from human (beta 1AR and alpha 2AR) and hamster (beta 2AR and alpha 1AR) were used to detect homologous sequences in other vertebrates, invertebrates and unicellular organisms by Southern blot hybridization analysis. Sequences homologous to hamster beta 2AR were detected in lower vertebrates, invertebrates and Dictyostelium, but not in yeast or bacteria. Within vertebrates, sequences strongly homologous to human beta 1AR and human platelet alpha 2AR were confined to the higher vertebrates only. In the invertebrates, only Drosophila contained sequences homologous to hamster alpha 1AR. Our results suggest that non-mammalian species may contain receptors homologous to the mammalian adrenergic receptors and that the sequences homologous to human beta 2AR have been the most strongly conserved.

The isolation and sequencing of a number of G protein-coupled receptors has now provided extensive primary structure information for this family of homologous proteins. The diverse nature of these receptors suggests that the family of proteins may grow to include receptors for many neurotransmitters and perhaps many peptide hormones. The topography of these receptors, a single polypeptide with seven transmembrane segments, appears to have features well suited for the transmission of signals, via conformational changes, to the interior of the cell. Detailed site-directed mutagenesis studies are now underway in many laboratories to understand the significance of the topography and also the regions of homology evident in the structures of all of these receptors. Obvious features of interest are the precise residues involved in the coupling of the receptors to the G-proteins and the identification of the residues required for ligand binding in each of the receptors, as well as domains of these receptors involved in the regulation of receptor function. In addition, the availability of molecular probes for this family of proteins will permit the elucidation of mechanisms of regulation at the gene level.

The major forms of beta-hexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) occur as multimers of alpha and beta chains--hexosaminidase A (alpha beta a beta b) and hexosaminidase B 2(beta a beta b). To facilitate the investigation of beta-chain biosynthesis and the nature of mutation in Sandhoff disease, a human hexosaminidase beta-chain cDNA clone was isolated. Hexosaminidase B (10 mg) was treated with CNBr, five peptide fragments were isolated by reverse-phase HPLC, and their amino acid sequences were determined. One of these contained a string of six amino acids from which an oligonucleotide probe was defined. The simian virus 40-transformed human fibroblast cDNA library of Okayama and Berg was screened by colony hybridization with the radiolabeled probe. Thirteen probe-binding clones were selected out of 50,000 clones screened. Four of these designated pHex were shown to be identical at their 3' ends by restriction enzyme mapping, differing only in their 5' extensions (1.4-1.7 kilobases). The nucleotide sequence of a 174-base-pair segment contained the deduced amino acid sequence of two of the five CNBr peptides, indicating that the pHex clones encode the beta subunit of hexosaminidase. In addition, pHex cDNA was found homologous to multiple bands in digests of genomic human DNA totaling 43 kilobases (kb), all of which were mapped to chromosome 5 in somatic cell hybrids, as expected of the HEXB gene. The pHex cDNA also hybridized to a 2.2-kilobase RNA that apparently codes for the pre-beta-polypeptide of hexosaminidase. This RNA species was absent in the fibroblasts of one of three patients with Sandhoff disease examined. We anticipate that these clones will be of value to diagnosis and carrier detection of Sandhoff disease in affected families.

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.

We report that a cysteine residue in the human beta 2-adrenergic receptor (beta 2AR) is covalently modified by thioesterification with palmitic acid. By site-directed mutagenesis of the receptor, we have identified Cys341 in the carboxyl tail of the protein as the most likely site of palmitoylation. Mutation of Cys341 to glycine results in a nonpalmitoylated form of the receptor that exhibits a drastically reduced ability to mediate isoproterenol stimulation of adenylyl cyclase. The functional impairment of this mutated beta 2AR is also reflected in a markedly reduced ability to form a guanyl nucleotide-sensitive high affinity state for agonists, characteristic of wild-type receptor. These results indicate that post-translational modification by palmitate of beta 2AR may play a crucial role in the normal coupling of the receptor to the adenylyl cyclase signal transduction system.

The lysosomal beta-hexosaminidases (N-acetyl-beta-glucosaminidase, EC 3.2.1.30) occur as two major isozymes, hexosaminidase A (alpha beta a beta b) and hexosaminidase B (2(beta a beta b)). To facilitate the investigations of the biosynthesis and structure of the enzymes and the nature of mutation in Tay-Sachs disease, we have isolated cDNA clones coding for the alpha-subunit. The polypeptide chains of hexosaminidase A (30 mg) were digested with trypsin, and peptides were isolated by reverse phase high pressure liquid chromatography and their amino acid sequences determined. One of alpha-chain peptides contained a string of seven amino acids from which two sets of oligonucleotides were specified. They were used to screen the SV40-transformed human fibroblast cDNA library of Okayama and Berg. Three cDNA clones, designated pHexA, identified from among 5 X 10(5) clones screened, contained the deduced amino-acid sequences of five alpha-chain peptides. Genomic DNA homologous to pHexA cDNA mapped to human chromosome 15 in somatic cell hybrids, as expected for the pre-alpha-polypeptide. Two of the clones contained identical polyadenylation sites, while the third was polyadenylated about 450 base pairs downstream. The two types of clones were found to correspond to a major 2.0-kilobase pair and a minor 2.3-kilobase pair mRNA species. Blot hybridizations of mRNA and DNA from Tay-Sachs variant fibroblasts revealed absence or reduction of levels of both mRNA species among infantile and juvenile variants, but no observable DNA alterations. Alignment of the pre-alpha- and pre-beta-polypeptides revealed 55% nucleotide and 57% amino acid homology. These data suggest a common origin of the HEXA and HEXB genes and account for the similar substrate specificities of the alpha-dimer subunit, hexosaminidase S, and hexosaminidase B.

We constructed and expressed in a permanent cell line a beta 2-adrenergic receptor with a valine substitution for cysteine 184 of the second putative extracellular loop. The mutant receptor was partially uncoupled from adenylyl cyclase with impaired ability to form the high affinity agonist-receptor-G protein complex, yet displayed more rapid and extensive agonist-induced desensitization. The enhanced desensitization was accompanied by increased agonist promoted, but not cAMP promoted, receptor phosphorylation in intact cells. Thus, not only is impaired desensitization associated with decreased phosphorylation, as we have shown with several mutant beta 2-adrenergic receptors recently, but enhanced desensitization is accompanied by increased agonist promoted receptor phosphorylation. In the case of this cysteine mutant, this may be due to the greater accessibility of the uncoupled receptor for phosphorylation by the beta-adrenergic receptor kinase.

Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.

We have studied cyclic AMP-mediated regulation of the beta 2-adrenergic receptor (beta 2AR). The effects of cAMP were assessed in Chinese hamster fibroblast (CHW) cells expressing either the wild type human beta 2AR receptor (CH-beta 2) or mutated forms of the receptor lacking the consensus sequences for phosphorylation by the cAMP-dependent protein kinase. Treatment of the CH-beta 2 cells with the cAMP analogue dibutyryl cAMP (Bt2cAMP) induces a time-dependent "down-regulation" of the number of beta 2AR. This down-regulation of the receptors is accompanied by a decline in the steady state level of beta 2AR mRNA. Moreover, the treatment with Bt2cAMP induces an increase in the phosphorylation level of the membrane-associated beta 2AR. Both the reduction in beta 2AR mRNA and the enhanced phosphorylation of the receptor are rapid and precede the loss of receptor. The down-regulation of beta 2AR induced by Bt2cAMP is concentration-dependent and mimicked by the other biologically active cyclic nucleotide analogue, 8-Br-cAMP, by forskolin, and by the phosphodiesterase inhibitor, isobutylmethylxanthine. In the CHW cell lines expressing receptors lacking the putative protein kinase A phosphorylation sites, the Bt2cAMP-induced phosphorylation of beta 2AR is completely abolished. In these cells the down-regulation of beta 2AR receptor number produced by cAMP is significantly slowed, whereas the reduction in beta 2AR mRNA level is equivalent to that observed in CH-beta 2 cells. These data indicate that there are at least two pathways by which cAMP may decrease the number of beta 2ARs in cells: one involves phosphorylation of the receptor by the cAMP-dependent protein kinase and the other leads to a reduction in steady state beta 2AR mRNA levels.

Eukaryotic cells have evolved a variety of mechanisms for dampening their responsiveness to hormonal stimulation in the face of sustained activation. The mechanisms for such processes, collectively referred to as desensitization, often involve alterations in the properties and number of cell-surface hormone receptors. It has been speculated that phosphorylation-dephosphorylation reactions, which are known to regulate the catalytic activities of enzymes, also regulate the function of receptors. Highly specific receptor kinases, such as rhodopsin kinase and beta-adrenergic receptor kinase, which show stimulus-dependent phosphorylation of receptors have been described. Direct evidence for a causal relationship between receptor phosphorylation and desensitization has been lacking however. Here we report that prevention of agonist-stimulated beta 2-adrenergic receptor (beta 2AR) phosphorylation by truncation of its serine and threonine-rich phosphate acceptor segment delays the onset of desensitization. We also show that selective replacement of these serine and threonine residues by alanine and glycine delays desensitization even further. These data provide the first direct evidence that one molecular mechanism of desensitization of G-protein-coupled receptors involves their agonist-induced phosphorylation.

The type and distribution of the oligosaccharides on each polypeptide of human placental beta-hexosaminidases A (alpha (beta a beta b)) and B (2(beta a beta b)) were examined. The denatured polypeptides were separated by isoelectric focussing in a polyacrylamide slab gel and each gel was then overlaid with 125I-labelled lectins. The study indicated that the beta a chain contains negligible carbohydrate, the beta b chain contains both the high-mannose and a complex type oligosaccharide, and the alpha chain contains predominantly high-mannose or hybrid type moieties. Two asparagine-linked high-mannose type oligosaccharides present on the beta b polypeptide of beta-hexosaminidase B were isolated by concanavalin A chromatography and by reverse-phase high pressure liquid chromatography. Proton nuclear magnetic resonance characterization of the oligosaccharides revealed an equimolar glycan mixture of the high-mannose type structure Man5 and Man6.

There are two major beta-hexosaminidase, EC 3.2.1.52, isozymes in normal human tissues. They exist as active dimers of alpha- and/or beta-subunits. A defect of their beta-subunit results in Sandhoff disease (O-variant GM2 gangliosidosis), an inherited, clinically heterogeneous, lysosomal storage disease. The status of the HEXB gene, pre beta-polypeptide chain mRNA, and residual beta-hexosaminidase activities were examined in a clinically and ethnically diverse collection of 16 fibroblast cell lines from patients with Sandhoff disease. Differentiation of the two major clinical types, infantile and juvenile onset, could be made by the determination of the activity of the residual beta-hexosaminidase eluting in the same pH range as hexosaminidase A. All the juvenile lines were found to have normal or reduced levels of pre beta-chain mRNA and no gross abnormalities in the HEXB gene. Of the 11 infantile type cell lines examined, four were found to contain no detectable pre beta-chain mRNA. Two cell lines in this group contained partial gene deletions localized to the 5' end of the HEXB gene. One of these cell lines has previously been assigned to the single complementation group in Sandhoff disease, conclusively demonstrating that the primary gene defect in the majority of Sandhoff cases is in the HEXB gene itself. These data suggest that each clinical group is made up of a collection of different HEXB mutations.

Continuous exposure of cells to neurotransmitter or hormone agonists often results in a rapid desensitization of the cellular response. For example, pretreatment of Chinese hamster fibroblasts (CHW cells) expressing beta 2-adrenergic receptors (beta 2AR) with low (nanomolar) concentrations of isoproterenol, a beta-adrenergic agonist, causes decreases in the sensitivity of the cellular adenylyl cyclase response to the agonist, without changing the maximal responsiveness. In contrast, exposure of CHW cells to high (micromolar) concentrations of isoproterenol results in decreases in both sensitivity and the maximal responsiveness to agonist. To explore the role(s) of receptor phosphorylation in these processes, we expressed in CHW cells three mutant beta 2AR genes encoding receptors lacking putative phosphorylation sites for the cAMP-dependent protein kinase A and/or the cAMP-independent beta 2AR kinase. Using these mutants we found that exposure of cells to low concentrations of agonist appears to preferentially induce phosphorylation at protein kinase A sites. This phosphorylation correlates with the decreased sensitivity to agonist stimulation of the adenylyl cyclase response. At higher agonist concentrations phosphorylation on both the beta 2AR kinase and protein kinase A sites occurs, and only then is the maximal cyclase responsiveness elicited by agonist reduced. We conclude that low or high concentrations of agonist elicit phosphorylation of beta 2AR on distinct domains, with different implications for the functional coupling of the receptors with effector molecules.