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J Hwa

Publications and source records attributed to J Hwa.

At least 19 recordsLinked to original sources
PubMed2001

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1.

A dark state tertiary structure in the cytoplasmic domain of rhodopsin is presumed to be the key to the restriction of binding of transducin and rhodopsin kinase to rhodopsin. Upon light-activation, this tertiary structure undergoes a conformational change to form a new structure, which is recognized by the above proteins and signal transduction is initiated. In this and the following paper in this issue [Cai, K., Klein-Seetharaman, J., Altenbach, C., Hubbell, W. L., and Khorana, H. G. (2001) Biochemistry 40, 12479-12485], we probe the dark state cytoplasmic domain structure in rhodopsin by investigating proximity between amino acids in different regions of the cytoplasmic face. The approach uses engineered pairs of cysteines at predetermined positions, which are tested for spontaneous formation of disulfide bonds between them, indicative of proximity between the original amino acids. Focusing here on proximity between the native cysteine at position 316 and engineered cysteines at amino acid positions 55-75 in the cytoplasmic sequence connecting helices I-II, disulfide bond formation was studied under strictly defined conditions and plotted as a function of the position of the variable cysteines. An absolute maximum was observed for position 65 with two additional relative maxima for cysteines at positions 61 and 68. The observed disulfide bond formation rates correlate well with proximity of these residues found in the crystal structure of rhodopsin in the dark. Modeling of the engineered cysteines in the crystal structure indicates that small but significant motions are required for productive disulfide bond formation. During these motions, secondary structure elements are retained as indicated by the lack of disulfide bond formation in cysteines that do not face toward Cys316 in the crystal structure model. Such motions may be important in light-induced conformational changes.

Amino Acid Sequence↗
PubMed2001

Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants.

Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110-Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.

Amino Acid Sequence↗
PubMed2001

Identification of potent and selective neuropeptide Y Y(1) receptor agonists with orexigenic activity in vivo.

Neuropeptide Y (NPY) binds to a family of G-protein coupled receptors termed Y(1), Y(2), Y(3), Y(4), Y(5), and y(6). The use of various receptor subtype-selective agonists and antagonists has facilitated identification of the receptor subtypes responsible for mediating many of the biological effects of NPY. For example, the potent orexigenic activity of NPY is believed to be mediated by both the Y(1) and Y(5) receptor subtypes. Several selective Y(5) receptor agonists that stimulate food intake in rodents are available, but no selective Y(1) receptor agonist has been reported. We have identified several NPY analogs that bind the NPY Y(1) receptor with high affinity and exhibit full agonist activity, measured as inhibition of forskolin-stimulated cAMP production in cells expressing the cloned NPY Y(1) receptor. [D-Arg(25)]-NPY, [D-His(26)]-NPY, Des-AA(10--17)[Cys(7,21),Pro(34)]-NPY, Des-AA(11--18)[Cys(7,21),D-Lys(9)(Ac)]-NPY, Des-AA(11--18)[Cys(7,21),D-Lys(9)(Ac),Pro(34)]-NPY, Des-AA(11--18)[Cys(7,21),D-Lys(9)(Ac),D-His(26)]-NPY and Des-AA(11--18)[Cys(7,21),D-Lys(9)(Ac),D-His(26), Pro(34)]-NPY bind the NPY Y(1) receptor with K(i) values of 0.9 +/- 0.2, 2.0 +/- 0.3, 0.2 +/- 0.05, 0.7 +/- 0.1, 0.2 +/- 0.01, 2.2 +/- 0.3, and 1.2 +/- 0.3 nM, respectively, and inhibit forskolin-stimulated cAMP production with EC(50) values of 0.2 +/- 0.02, 0.5 +/- 0.04, 0.3 +/- 0.03, 0.5 +/- 0.05, 0.4 +/- 0.16, 5.3 +/- 0.32, and 5.1 +/- 0.97 nM, respectively. These peptides are highly selective for the NPY Y(1) receptor relative to the NPY Y(2), Y(4), and Y(5) receptors. [D-Arg(25)]-NPY, [D-His(26)]-NPY and Des-AA(11--18)[Cys(7,21), D-Lys(9)(Ac),D-His(26),Pro(34)]-NPY stimulate food intake dose-responsively in Long-Evans rats for at least 4 h after intracerebroventricular administration. Although the involvement of Y(1) receptors in several physiological activities, such as vasoconstriction and anxiolysis, remains to be investigated, adequate tools are now available.

Animals↗
PubMed2000

Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation.

alpha(1)-adrenergic receptors (alpha(1)-ARs) are members of the G-protein-coupled receptor (GPCR) superfamily and activate inositol phosphate (IP) turnover. We show that glycine and asparagine mutations of Phe303 in transmembrane segment VI (TMVI) of the alpha(1B)-AR, a highly conserved residue in GPCRs, although increasing agonist affinity, abolish agonist-activated IP signalling. Co-expression of the Phe303 mutants also inhibited (-)epinephrine-stimulated IP signalling by wild-type alpha(1B)-AR and other G(q)-coupled receptors, as well as IP signalling mediated by AlF(4)(-) stimulation of both wild-type G(q alpha) and a constitutively active mutant. The inability of the Phe303 mutants to signal is due to induction of a receptor conformation that dissociates G-protein binding from activation. As a result, the Phe303 mutants sequester G(q alpha) and stoichiometrically inhibit Gq signalling in a dominant-negative manner. We further show that both the enhanced basal and agonist-stimulated IP-signalling activity of the constitutively active alpha(1B)-AR mutants, C128F and A293E, are inhibited in the double mutants, C128F/F303G and A293E/F303G. Phe303, therefore, appears to be critically involved in coupling TMVI alpha-helical movement, a key step in receptor activation, to activation of the cognate G-protein.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1999

Structure and function in rhodopsin: effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase.

Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end of helix III) and Cys250 (end of helix VI); disulfide bond 5, between Cys135 (end of helix III) and Cys250 (end of helix VI); and disulfide bond 6, between Cys245 (end of helix VI) and Cys338 (C-terminus). The effects of local restrictions caused by the cross-links on transducin (G(T)) activation and phosphorylation by rhodopsin kinase (RK) following illumination were studied. Disulfide bond 1 showed little effect on either G(T) activation or phosphorylation by RK, suggesting that the relative motion between interhelical loop I-II and helix VII is not crucial for recognition by G(T) or by RK. In contrast, disulfide bonds 2-5 abolished both G(T) activation and phosphorylation by RK. Disulfide bond 6 resulted in enhanced G(T) activation but abolished phosphorylation by RK, suggesting the structure recognized by G(T) was stabilized in this mutant by cross-linking of the C-terminus to the cytoplasmic end of helix VI. Thus, the consequences of the disulfide cross-links depended on the location of the restriction. In particular, relative motions of helix VI, with respect to both helices III and VII upon light activation, are required for recognition of rhodopsin by both G(T) and RK. Further, the conformational changes in the cytoplasmic face that are necessary for protein-protein interactions need not be cooperative, and may be segmental.

Amino Acid Sequence↗
PubMed1999

Single-cysteine substitution mutants at amino acid positions 55-75, the sequence connecting the cytoplasmic ends of helices I and II in rhodopsin: reactivity of the sulfhydryl groups and their derivatives identifies a tertiary structure that changes upon light-activation.

Cysteines were introduced, one at a time, at amino acid positions 55-75 in the cytoplasmic region connecting helices I and II in rhodopsin. In each of the 21 cysteine mutants, the reactive native cysteine residues (C140 and C316) were replaced by serine. Except for N55C, all mutants formed rhodopsin-like chromophores and had normal photobleaching characteristics. The efficiency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The reactivity of the substituted cysteine in each mutant toward 4, 4'-dithiodipyridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and I75C were unreactive to 4-PDS under the conditions used, suggesting that they are embedded in the micelle or protein interior. The mutants V63C, H65C-T70C, and N73C reacted rapidly, while the remainder of the mutants reacted more slowly, and varied in reactivity with sequence position. For the mutants derivatized with 4-PDS, the rate of release of thiopyridone from the resulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investigated in the dark and in the light. Marked changes in the rates of thiopyridone release in the light were found at specific sites. Collectively, the data reveal tertiary interactions of the residues in the sequence investigated and demonstrate structural changes due to photoactivation.

Amino Acid Sequence↗
PubMed1999

Structural features and light-dependent changes in the sequence 59-75 connecting helices I and II in rhodopsin: a site-directed spin-labeling study.

Twenty-one single-cysteine substitution mutants were prepared in the sequence 56-75 between transmembrane helices I and II at the cytoplasmic surface of bovine rhodopsin. Each mutant was reacted with a sulfhydryl-specific reagent to produce a nitroxide side chain. The electron paramagnetic resonance of the labeled proteins in dodecyl maltoside solution was analyzed to provide the relative mobility and accessibility of the nitroxide side chain to both polar and nonpolar paramagnetic reagents. The results indicate that the hydrophobic-water interface of the micelle intersects helices I and II near residues 64 and 71, respectively. Thus, the sequence 64-71 is in the aqueous phase, while 56-63 and 72-75 lie in the transmembrane helices I and II, respectively. The lipid-facing surfaces on transmembrane helices I and II near the cytoplasmic surface correspond to approximately 180 degrees and 90 degrees of arc on the helical surfaces, respectively. Photoactivation of rhodopsin produced changes in structure in the region investigated, primarily around helix II. However, these changes are much smaller than those noted by spin labels in helix VI (Altenbach, C., Yang, K., Farrens, D., Farahbakhsh, Z., Khorana, H. G., and Hubbell, W. L. (1996) Biochemistry 35, 12470).

Amino Acid Sequence↗
PubMed1999

Structure and function in rhodopsin: kinetic studies of retinal binding to purified opsin mutants in defined phospholipid-detergent mixtures serve as probes of the retinal binding pocket.

In the current standard procedure for preparation of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 microM 11-cis-retinal before detergent solubilization for purification. We found that binding of 11-cis-retinal to opsin mutants with single amino acid changes at Trp-265 (W265F,Y,A) and a retinitis pigmentosa mutant (A164V) was far from complete and required much higher concentrations of 11-cis-retinal. By isolation of the expressed opsins in a stable form, kinetic studies of retinal binding to the opsins in vitro have been carried out by using defined phospholipid-detergent mixtures. The results show wide variation in the rates of 11-cis-retinal binding. Thus, the in vitro reconstitution procedure serves as a probe of the retinal-binding pocket in the opsins. Further, a method is described for purification and characterization of the rhodopsin mutants after retinal binding to the opsins in vitro.

Amino Acid Sequence↗
PubMed1999

Structure and function in rhodopsin: further elucidation of the role of the intradiscal cysteines, Cys-110, -185, and -187, in rhodopsin folding and function.

The disulfide bond between Cys-110 and Cys-187 in the intradiscal domain is required for correct folding in vivo and function of mammalian rhodopsin. Misfolding in rhodopsin, characterized by the loss of ability to bind 11-cis-retinal, has been shown to be caused by an intradiscal disulfide bond different from the above native disulfide bond. Further, naturally occurring single mutations of the intradiscal cysteines (C110F, C110Y, and C187Y) are associated with retinitis pigmentosa (RP). To elucidate further the role of every one of the three intradiscal cysteines, mutants containing single-cysteine replacements by alanine residues and the above three RP mutants have been studied. We find that C110A, C110F, and C110Y all form a disulfide bond between C185 and C187 and cause loss of retinal binding. C185A allows the formation of a C110-C187 disulfide bond, with wild-type-like rhodopsin phenotype. C187A forms a disulfide bond between C110 and C185 and binds retinal, and the pigment formed has markedly altered bleaching behavior. However, the opsin from the RP mutant C187Y forms no rhodopsin chromophore.

Amino Acid Sequence↗
PubMed1997

Structure and function in rhodopsin: packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain are coupled.

A previous study of the retinitis pigmentosa mutation L125R and two designed mutations at this site, L125A and L125F, showed that these mutations cause partial or total misfolding of the opsins expressed in COS cells from the corresponding mutant opsin genes. We now report on expression and characterization of the opsins from the following retinitis pigmentosa mutants in the transmembrane domain of rhodopsin that correspond to six of the seven helices: G51A and G51V (helix A), G89D (helix B), A164V (helix D), H211P (helix E), P267L and P267R (helix F), and T297R (helix G). All the mutations caused partial misfolding of the opsins as observed by the UV/visible absorption characteristics and by separation of the expressed opsins into fractions that bound 11-cis-retinal to form the corresponding mutant rhodopsins and those that did not bind 11-cis-retinal. Further, all the mutant rhodopsins prepared from the above mutants, except for G51A, showed strikingly abnormal bleaching behavior with abnormal metarhodopsin II photointermediates. The results show that retinitis pigmentosa mutations in every one of the transmembrane helices can cause misfolding of the opsin. Therefore, on the basis of these and previous results, we conclude that defects in the packing of the transmembrane helices resulting from these mutations are relayed to the intradiscal domain, where they cause misfolding of the opsin by inducing the formation of a disulfide bond other than the native Cys-110---Cys-187 disulfide bond. Thus, there is coupling between packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain.

Amino Acid Sequence↗
PubMed1997

Synergism of constitutive activity in alpha 1-adrenergic receptor activation.

Recently a number of mutations have been found in vitro which maintain alpha 1-adrenergic receptors (ARs) in a partially activated form. We have previously identified two amino acid residue positions in the alpha 1b-adrenergic receptor (AR), Cys128 and Ala204, one in each of the third and fifth transmembrane segments, that constitutively activate the receptor when substituted for a phenylalanine or valine, respectively [Perez et al. (1996) Mol. Pharmacol. 49, 112-122; Hwa et al. (1996) J. Biol. Chem. 271, 7956-7964]. Another mutation analyzed previously, Ala293Glu, located in the third intracellular loop, also constitutively activates the receptor [Kjelsborg et al. (1992) J. Biol. Chem. 267, 1430-1433]. All three mutations displayed similar manifestations of constitutive activity such as higher binding affinity and potency for agonists as well as higher basal signal transduction as predicted by the revised ternary complex model of receptor activation. We hypothesized that the individual mutations because of their critical location alter the conformation of the transmembrane helices such that mimicry occur that partially conforms to the activated state, R*. To explore whether these potential conformations are independent, we combined these three mutations in all possible permutations. The combined triple mutation displays 700-fold higher binding affinity for (-)-epinephrine and 20-fold higher basal IP3 release than the wild-type receptor. We also observed that each mutation contributed independently and synergistically to both receptor agonist binding and activation with the combined mutations basal activity exceeding that of the fully-stimulated wild-type receptor. There was also a direct correlation between epinephrine's binding affinity and the degree of constitutive activity. Because the mutations affect different transmembrane domains, these results are consistent with a mechanism that helical movement acts in a concerted fashion in agonist-induced activation, a synergism predicted if multiple helix movement is involved in receptor activation.

Animals↗
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

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↗