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D M Perez

Publications and source records attributed to D M Perez.

36 records · Page 2Linked to original sources
PubMed1996

Activation of the alpha1b-adrenergic receptor is initiated by disruption of an interhelical salt bridge constraint.

Rhodopsin receptor activation involves the disruption of a salt bridge constraint between glutamic acid 113 on transmembrane 3 and a lysine 296 in transmembrane 7, which forms a Schiff's base with retinal. Light-induced isomerization of cis-retinal to the all trans form breaks this rhodopsin salt bridge leading to receptor activation. The analogous residues in alpha1b-adrenergic receptors, aspartic acid 125 and lysine 331, also have the potential of forming a constraining salt bridge holding the receptor to an inactive protein configuration. This alpha1b-adrenergic receptor salt bridge constraint is then released upon binding by the receptor agonist. To test this hypothesis, site-directed mutagenesis was used to eliminate the positive charge at position 331 by substitution of an alanine. The expressed alpha1b-adrenergic receptor mutant demonstrated a 6-fold increased epinephrine binding affinity with no alterations of affinity values for selective adrenergic receptor antagonists. Furthermore, an increased epinephrine potency for total soluble inositol phosphate production along with an elevated basal inositol triphosphate level was observed in COS-1 cells transfected with mutant versus wild-type alpha1b-adrenergic receptors. Similar results were obtained for a lysine to a glutamic acid alpha1b-adrenergic receptor mutation. In addition, increased basal inositol triphosphate levels were also observed for two aspartic acid 125 alpha1b-adrenergic receptor mutations, consistent with this residue's role as the counterion of the salt bridge. Taken together, these alpha1b-adrenergic receptor mutations suggest a molecular mechanism by which the positively charged lysine 331 stabilizes the negatively charged aspartic acid 125 via a salt bridge constraint until bound by the receptor agonist.

Animals↗
PubMed1996

Chimeras of alpha1-adrenergic receptor subtypes identify critical residues that modulate active state isomerization.

We have identified previously two amino acids, one in each of the fifth and sixth transmembrane segments of both the alpha1a-adrenergic receptor and the alpha1b-adrenergic receptor (AR), that account almost entirely for the selectivity of agonist binding by these receptor subtypes (Hwa, J., Graham, R. M., and Perez, D. M. (1995) J. Biol. Chem. 270, 23189-23195). Thus reversal of these two residues, from those found in the native receptor of one subtype to those in the other subtype, produces complementary changes in subtype selectivity of agonist binding. Here we show that mutating only one of these residues in either the alpha1b-AR or the alpha1a-AR to the corresponding residue in the other subtype (Ala204 --> Val for the alpha1b; Met292 --> Leu for the alpha1a-AR) results in chimeras that are constitutively active for signaling by both the phospholipase C and phospholipase A2 pathways. This is evident by an increased affinity for agonists, increased basal phospholipase C and phospholipase A2 activation, and increased agonist potency. Although mutation of the other residue involved in agonist binding selectivity, to the corresponding residue in the other subtype (Leu314 --> Met for the alpha1b-AR; Val185 --> Ala for the alpha1a-AR) does not alter receptor binding or signaling, per se, when combined with the corresponding constitutively activating mutations, the resulting chimeras, Ala204 --> Val/Leu314 --> Met ( alpha1b-AR) and Val185 --> Ala/Met292 --> Leu ( alpha1a-AR), display wild type ligand binding and signaling. A simple interpretation of these results is that the alpha1a- and alpha1b-ARs possess residues that critically modulate isomerization from the basal state, R, to the active state R*, and that the native receptor structures have evolved to select residues that repress active state isomerization. It is likely that the residues identified here modulate important interhelical interactions between the fifth and sixth transmembrane segments that inhibit or promote receptor signaling.

Animals↗
PubMed1996

The unique nature of the serine interactions for alpha 1-adrenergic receptor agonist binding and activation.

Activation of the beta2- and alpha2-adrenergic receptors (AR) involves hydrogen bonding of serine residues in the fifth transmembrane segment (TMV) to the catechol hydroxyls of the endogenous agonists, epinephrine and norepinephrine. With the beta2-AR both Ser204 and Ser207 but not a third TMV serine (Ser203) are required for binding and full agonist activity. However, with the alpha2a-AR only one of two TMV serines (Ser204, equivalent to Ser207 in the beta-AR) appears to contribute partially to agonist-binding and activation. Because the alpha1a-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in alpha1-AR activation. Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine- substitution mutants that lacked either Ser188 (Ser188--> Ala) or Ser192 (Ser192-->Ala). With the substitution of both serines in the double mutant, Ser188/192-->Ala, binding of all three ligands was significantly reduced (10- 100-fold) consistent with a single hydrogen bond interaction. However, receptor-mediated inositol phosphate production was markedly attenuated only with the Ser188-->Ala mutation and not with Ser192-->Ala. In support of the importance of Ser188, binding of phenylephrine (meta-hydroxyl only) by Ser192-->Ala increased 7-fold over that observed with either the wild type receptor or the Ser188-->Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser192-->Ala mutation. In addition, when combined with a recently described constitutively active alpha1a-AR mutation (Met292-->Leu), only the Ser188-->Ala mutation and not Ser192-->Ala relieved the high affinity binding and increased agonist potency observed with the Met292-->Leu mutation. A simple interpretation of these findings is that the meta-hydroxyl of the endogenous agonists preferentially binds to Ser188, and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser192, that allows receptor activation. Furthermore, since Ser188 and Ser192 are separated by three residues on the TMV alpha-helix, whereas Ser204 and Ser207 of the beta2-AR are separated by only two residues, the orientation of the catechol ring in the alpha1-AR binding pocket appears to be unique and rotated approximately 120 degrees to that in the beta2-AR.

Adrenergic alpha-Agonists↗
PubMed1996

Recent advances in the molecular pharmacology of the alpha 1-adrenergic receptors.

This review is intended to discuss recent developments in the molecular pharmacology of the alpha 1-adrenergic receptor (alpha 1-AR) subtypes. After a brief historical development, we will focus on the more contemporary issues having to do with this receptor family. Emphasis will be put on recent data regarding the cloning, nomenclature, signalling mechanisms, and genomic organization of the alpha 1-AR subtypes. We will also highlight recent mutational studies that identify key amino acid residues involved in ligand binding, as well as the role of the alpha 1-AR subtypes in regulating physiologic processes.

Aging↗
PubMed1996

Constitutive activation of a single effector pathway: evidence for multiple activation states of a G protein-coupled receptor.

A cysteine-to-phenylalanine mutation in the third transmembrane domain of the alpha 1B-adrenergic receptor constitutively activates the receptor, resulting in G protein coupling in the absence of agonist and activation of only a single effector pathway (phospholipase C but not phospholipase A2). This mutant receptor displays a higher affinity for the catecholamines, norepinephrine, and epinephrine, as well as for other phenethylamines, but not for imidazolines, a class of structurally distinct alpha agonists. Dose-response studies demonstrate a higher potency and intrinsic activity of phenethylamines for polyphosphoinositide turnover but not for arachidonic acid release. Imidazolines have wild-type potencies and intrinsic activities for both pathways. These data indicate that a single receptor subtype forms multiple conformations (i.e., exhibits induced conformational pleiotropy) for G protein interactions (high affinity states) that are specific for a particular G protein/effector pathway and that multiple binding sites exist for agonists, which promote or induce these specific interactions. Pharmacological diversity may, thus, be achieved through a single receptor by the development of compounds that induce a single activated conformer. This has major ramifications for the eventual development of signaling-specific therapeutics.

Adrenergic alpha-Antagonists↗
PubMed1996

Identification of critical extracellular loop residues involved in alpha 1-adrenergic receptor subtype-selective antagonist binding.

alpha 1-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. Although structurally similar, the three cloned subtypes (alpha 1a-AR, alpha 1b-AR, and alpha 1d-AR) bind a series of ligands with different relative potencies. This is particularly true for the alpha 1a-AR, which recognizes a number of ligands with 10-100-fold higher affinity than the alpha 1b or alpha 1d subtypes. Because ligands are hypothesized to bind to receptor residues that are located in the transmembrane (TM) spanning domains, subtype differences in ligand recognition are likely the result of differences in the binding properties of nonconserved TM residues. We previously reported on the identification of two TM residues in the alpha 1b-AR that converted the agonist binding profile entirely to that of the alpha 1a-AR when mutated to corresponding alpha 1a-AR residues. We now report on the determinants of antagonist selectivity between these two alpha 1-AR subtypes. Construction of a chimera in which the entire fifth TM and a portion of the putative second extracellular loop of the hamster alpha 1b-AR was replaced with the corresponding region of the rat alpha 1a-AR revealed that the chimera accounted for all of the higher binding affinity (8-29-fold) seen in the alpha 1a-AR for two antagonists, phentolamine and WB4101. Using site-directed mutagenesis, we further analyzed individual point mutations making up this chimera. We found that three adjacent residues, which were located on the extracellular loop of the fifth TM, are fully responsible for this higher antagonist binding affinity in the alpha 1a-AR. These three point mutations (G196Q, V1971, T198N) in the alpha 1b-AR were additive and sufficient in their effects on changing antagonist-binding profiles to that of the alpha 1a-AR. Reversal of these three residues in the alpha 1a-AR to their corresponding residues in the alpha 1b-AR completely reversed the antagonist affinity to wild-type alpha 1b-AR values. To aid in molecular modeling, the use of organic chemicals that mimic key structures of the antagonists were used in competitive ligand-binding studies with the mutated receptors. These results indicated the orientation of both phentolamine and WB4101 in the alpha 1-AR binding pocket. Together, the data indicate that alpha 1-antagonists may bind near the surface of the receptor, much like the peptide hormone receptors, and not deep within the TM regions, where the ligand-binding pocket was first proposed and identified for alpha 1 agonists.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1995

Identification of critical determinants of alpha 1-adrenergic receptor subtype selective agonist binding.

alpha 1-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. The three cloned subtypes (alpha 1a-AR, alpha 1b-AR, alpha 1d-AR), although structurally similar, bind a series of ligands with different relative potencies. This is particularly true for the alpha 1a-AR, which recognizes a number of agonists and antagonists with 5-50-fold higher affinity than the alpha 1b- or alpha 1d- subtypes. Since ligands bind to receptor-residues that are located in the transmembrane spanning domains, we hypothesize that subtype differences in ligand recognition are due to differences in the binding properties of nonconserved transmembrane residues. Using site-directed mutagenesis, selected putative ligand-binding residues in the alpha 1b-AR were converted, either individually or in combination, to the corresponding residues in the alpha 1a-AR. Mutation of two such residues (of approximately 172 amino acids in the transmembrane domains) converted the agonist binding profile entirely to that of the alpha 1a-AR. Over 80% of this conversion was due to an Ala204-->Val substitution; the remainder was due to the additional substitution of Leu314-->Met. To confirm that Ala204 and Leu314 are indeed critical for agonist subtype-selectivity, the equivalent residues in the alpha 1a-AR (Val185 and Met293) were reversed of that of the alpha 1b-AR. Correspondingly, the agonist-binding profile of this double alpha 1a-AR mutant reverted to that of the alpha 1b-AR. From these data, in conjunction with macromolecular modeling of the ligand-binding pocket, a model has been developed, which indicates that the determinants of these two residues for agonist subtype-selectivity are due not only to interactions between their side chains and specific ligand moieties but also to a critical interaction between these two amino acids.

Adrenergic alpha-Agonists↗
PubMed1995

The specific contribution of the novel alpha-1D adrenoceptor to the contraction of vascular smooth muscle.

With a selective antagonist, the specific contribution of the alpha-1D adrenoceptor (AR) to vascular smooth muscle contraction has been assessed. BMY 7378 bound to membranes expressing the cloned rat alpha-1D AR with a > 100-fold higher affinity (K1 = 2 nM) than binding to either the cloned rat alpha-1A AR (Ki = 800 nM) or the hamster alpha-1B AR (Ki = 600 nM). BMY 7378 exhibited differential potency in inhibiting vascular smooth muscle contraction. In the rat aorta and iliac artery, BMY 7378 was a high-affinity antagonist, producing parallel shifts in the phenylephrine concentration-response curve. The dissociation constants for this compound by Schild analysis were 0.95 and 4 nM for the aorta and iliac artery, respectively. The slopes of these Schild plots were not significantly different from unity. BMY 7378 was a weak antagonist in the rat caudal, mesenteric resistance and renal arteries, with Schild slopes significantly < 1. With ribonuclease protection assays, alpha-1D mRNA was found in all blood vessels examined. These data suggest that (1) BMY 7378 is a selective alpha-1D AR antagonist that can be used in functional systems to assess the contribution of this receptor in vascular smooth muscle contraction; (2) the alpha-1D AR appears to play a major role in the contraction of the aorta and iliac artery; (3) despite the fact that the mRNA for the alpha-1D AR can be detected in the caudal, mesenteric resistance (4) and renal arteries, it does not appear to play a role in mediating contraction of these blood vessels; and (4) expression of alpha-1D mRNA in a particular artery does not ensure that this receptor is involved in regulating the contraction of that artery.

Animals↗
PubMed1994

Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function.

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.

Amino Acid Sequence↗
PubMed1994

Identification of the mRNA for the novel alpha 1D-adrenoceptor and two other alpha 1-adrenoceptors in vascular smooth muscle.

In situ hybridization histochemistry, radioligand binding, and in vitro contractile studies were used to characterize the vascular distribution of the recently discovered alpha 1D-adrenoceptor. In situ hybridization with an antisense probe localized the mRNA for the alpha 1D-adrenoceptor to the medial layer of the rat aorta renal and mesenteric resistance arteries. If the tissues were first treated with RNase or a sense probe was used, no specific hybridization signal was detected. The extent to which this receptor was expressed as protein was assessed with radioligand binding studies. A series of ligands used to characterize alpha 1-adrenoceptors interacted with two sites labeled by [3H]prazosin in homogenates from the aorta and mesenteric vascular bed. The high affinity site had the characteristics of an alpha 1A-adrenoceptor. However, the low affinity site had ligand binding characteristics distinct from those of the alpha 1D-adrenoceptor or any other known alpha 1-adrenoceptor subtype. mRNA for the alpha 1B- and alpha 1C-adrenoceptors was also detected in the aorta, renal arteries, and mesenteric resistance arteries. Chloroethylclonidine (CEC) (1 and 10 microM) had differential effects on phenylephrine-induced contractions of vascular smooth muscle. CEC completely inhibited the response in the aorta and caused a partial inhibition in the mesenteric resistance artery. The same concentrations of CEC had little effect on phenylephrine responses in the renal artery. The data suggest the following. 1) mRNA for the novel alpha 1D-adrenoceptor is localized in vascular smooth muscle. 2) Definitive identification of expression of this receptor was not possible; this may be related to coexpression of other subtypes of alpha 1-adrenoceptors. 3) mRNA for the alpha 1C-adrenoceptor was detected in rat peripheral vasculature. 4) The alpha 1A-adrenoceptor is also localized in the vasculature. 5) Three and possibly four alpha 1-adrenoceptors participate in vascular smooth muscle regulation.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1994

Cloning, expression, and tissue distribution of the rat homolog of the bovine alpha 1C-adrenergic receptor provide evidence for its classification as the alpha 1A subtype.

Three alpha 1-adrenergic receptors (ARs) have been cloned, i.e., the alpha 1B-, alpha 1C-, and alpha 1D-ARs. Compared with the alpha 1B subtype, the alpha 1A subtype in tissue is described as being insensitive to chloroethylclonidine and sensitive to SZL-49 and having a 10-100-fold higher affinity for a number of agonists and antagonists. The alpha 1A subtype is also expressed in a variety of rat tissues (as assessed by pharmacology), with greatest abundance in the cerebral cortex, hippocampus, vas deferens, and submaxillary gland. The cloned bovine alpha 1C-AR, though having an alpha 1A-AR pharmacology, was first reported as not being expressed in any rat tissue (as determined by Northern analysis) and was therefore designated as a new subtype. We report the cloning, expression, and characterization of the rat homolog of the bovine alpha 1C-AR. Using a human alpha 1C-AR probe obtained by polymerase chain reaction screening of a neuroblastoma cell line (SK-N-MC), both exon 1 and exon 2 of the rat alpha 1C-AR gene were cloned from a rat genomic library. These two exons were spliced together and cloned into the expression vector pMT2'. Transfection into COS-1 cells and analysis of the ligand-binding profile of the expressed protein receptor using 125I-HEAT revealed a 10-100-fold higher affinity for the alpha 1-AR antagonists 5-methylurapidil, (+)-niguldipine, WB-4101, and phentolamine and the agonists oxymetazoline and methoxamine, compared with the alpha 1B-AR. This ligand-binding profile is similar to that for endogenously expressed tissue alpha 1A-ARs. In addition, the rat alpha 1C-AR was the least sensitive of the three cloned subtypes to the alkylating effects of chloroethylclonidine but was the most sensitive to the alkylating prazosin analog SZL-49, properties also observed for the tissue alpha 1A subtype. Furthermore, by three different techniques, i.e., RNase protection assays, reverse transcription-polymerase chain reaction Northern blotting, and in situ hybridization histochemistry, the rat alpha 1C-AR mRNA was localized to alpha 1A-AR-rich tissues, such as rat vas deferens, hippocampus, aorta, and submaxillary gland. Taken together, these data suggest that this receptor may actually represent the alpha 1A subtype.

Amino Acid Sequence↗
PubMed1993

Coupling of expressed alpha 1B- and alpha 1D-adrenergic receptor to multiple signaling pathways is both G protein and cell type specific.

alpha 1-Adrenergic receptors (ARs) are members of the G protein-coupled receptor superfamily. alpha 1-AR subtypes mediate the effects of the sympathetic nervous system, especially those involved in cardiac homeostasis. To investigate signal transduction by a novel subtype (alpha 1D), which we recently cloned, and to compare it with that by the previously characterized alpha 1B-AR, we assessed the ability of each subtype to activate polyphosphoinositide (PI) metabolism, cAMP accumulation, and arachidonic acid release in Chinese hamster ovary (CHO) and COS-1 cells expressing these subtypes after stable or transient transfection, respectively. In COS-1 and CHO cells, both the alpha 1D- and alpha 1B-AR were found to couple to PI hydrolysis through a pertussis toxin-insensitive G protein. Both alpha 1-AR subtypes also increased intracellular cAMP by an indirect mechanism, although this effect was observed only in COS-1 cells and not in CHO cells. Interestingly, alpha 1-AR-stimulated arachidonic acid release was also demonstrated for both subtypes in COS-1 cells. This release was mediated through phospholipase A2 activation and involved a pertussis toxin-sensitive G protein. alpha 1-AR-stimulated arachidonic acid release was dependent upon extracellular calcium and was inhibited by 1 microM nifedipine. Inhibitors of protein kinase C, phospholipase C, and diacylglycerol lipase did not alter alpha 1-AR-stimulated release of arachidonic acid. These findings indicate that in COS-1 cells alpha 1-AR-stimulated arachidonic acid release is most likely coupled to dihydropyridine-sensitive L-type calcium channels via a pertussis toxin-sensitive G protein. The influx of extracellular calcium then stimulates phospholipase A2 to release arachidonic acid. alpha 1-AR-stimulated arachidonic acid release could also be demonstrated in CHO cells and was pertussis toxin sensitive but nifedipine insensitive. These cells were also unresponsive to Bay K8644, indicating a lack of voltage-sensitive calcium channels in CHO cells. Nevertheless, alpha 1-AR activation increased intracellular Ca2+ levels, as assessed by fura-2 fluorescence studies. Neomycin blocked both alpha 1-AR-stimulated PI hydrolysis and increases in intracellular Ca2+ levels but did not inhibit the increase in arachidonic acid release. Taken together, these data indicate that in CHO cells alpha 1-ARs can couple directly to phospholipase A2 activation via a pertussis toxin-sensitive pathway. Thus, in these model systems we demonstrate for the first time that a single alpha 1-AR subtype can activate multiple distinct signal transduction pathways, in which receptor-effector coupling is modulated by distinct G proteins.

Animals↗
PubMed1992

Genomic organization and expression of the human alpha 1B-adrenergic receptor.

alpha 1-Adrenergic receptors (ARs) are members of the guanine nucleotide-binding protein-coupled receptor superfamily. The genes for all ARs described thus far are intronless. We report here the cloning and the nucleotide sequence of the gene for the human alpha 1B-AR. It consists of two exons and a single large intron of at least 20 kilobases which interrupts the coding region at the end of the putative sixth transmembrane domain. The deduced amino acid sequence of the encoded receptor has a high degree of homology to the cloned hamster, rat, and dog alpha 1B-ARs. To characterize the encoded protein, a fusion gene constructed by splicing together exon 1 and exon 2 was expressed transiently in COS-1 cells. The transfected gene fusion product resulted in the production of an alpha 1B-AR with ligand binding characteristics indistinguishable from those of the expressed hamster alpha 1B cDNA. Evidence that the human alpha 1B-AR gene we have isolated is indeed transcribed is the finding of similar sized (2.8-kilobase) transcripts in human heart and other tissues by Northern blot analysis when either exon 1 or exon 2 is used as a probe. Moreover, using primers designed to span the exon 1/exon 2 boundary, a polymerase chain reaction product generated from single-stranded DNA prepared from human heart mRNA had the exact size and nucleotide sequence predicted for a transcript in which exon 1 is spliced to exon 2. The 5'-flanking region (924 base pairs (bp)) of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content (70%) and contains several Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 5'-untranslated region also contains a putative cyclic AMP response element. Primer extension studies and RNase protection assays suggested that there are several potential transcription start sites in most tissues with a predominant site located 173 bp upstream from the translation start site. The 3'-flanking region contains a putative polyadenylation signal (ATTAAA) 492 bp downstream from the stop codon. The genomic organization of the human alpha 1B-AR with a single large intron interrupting its coding region differs from those of other ARs as well as muscarinic and 5-hydroxy-tryptamine receptors, which are intronless. The location of the intron in the human alpha 1B-AR gene is also unique among those members of the G-protein-coupled receptor family that do possess introns.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Cloning of the gene and cDNA for human heart chymase.

We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.

Amino Acid Sequence↗
PubMed1991

Solution-phase library screening for the identification of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA.

alpha 1-Adrenergic receptor (alpha 1-AR) subtypes (alpha 1A and alpha 1B) play a critical role in vascular smooth muscle contraction and circulatory homeostasis. Transcripts for these guanine nucleotide-binding protein-coupled receptors are extremely low in abundance, however, and isolation of their cDNAs is difficult. We have developed a novel technique for identifying rare clones in a cDNA library, which has been used successfully to isolate a cDNA clone encoding an alpha 1D-AR. A 564-bp polymerase chain reaction product encoding a region between the third and sixth transmembrane domains of the alpha 1D-AR was first generated using rat brain mRNA as template and highly degenerate primers. The primers corresponded to those domains but contained mismatches to the alpha 1B-AR sequences. A 3-kb transcript was identified with this polymerase chain reaction probe, by Northern analysis of rat hippocampus. However, traditional plaque hybridization failed to identify a cDNA in a rat hippocampus lambda gt10 library. By solution-phase screening of virtually the entire library, a cDNA containing a 3-kb insert was identified, amplified, and purified. This insert encodes a 560-amino acid protein corresponding to the topology of guanine nucleotide-binding protein-coupled receptors. This receptor has approximately 71% amino acid identity, in the transmembrane regions, to the hamster and rat alpha 1B-ARs. Characterization of the receptor expressed in COS-7 cells, by ligand binding and photoaffinity labeling, revealed some of the characteristics of an alpha 1A-AR. However, unlike alpha 1A-ARs characterized previously in membrane preparations or in solubilized partially purified preparations, the expressed receptor could be extensively inactivated by chlorethylclonidine. In addition, it displays ligand-binding properties that are not consistent with an alpha 1A-AR. This indicates that the cDNA clone that we have isolated encodes a novel alpha 1-AR subtype, which we classify as the alpha 1D-AR.

Animals↗
PubMed1980

The influence of utterance length upon bilabial closure duration of selected deaf children.

Measurements were made of intraoral air pressure associated with bilabial closure durations of VCV syllables in sentence-like frames of increasing length for nine deaf and nine matched normal hearing chidren. Results demonstrated significantly longer absolute closure durations at all utterance lengths for the deaf, but with the same relative decrease in closure durations with increases in utterance length for both groups. The deaf children showed lower peak intraoral air pressure values with more variability than the normal children. In addition, the deaf children had longer air-pressure pulse durations than did the normals. Clinical application of these findings to speech management are discussed.

Articulation Disorders↗