RESUMEN
The G protein-coupled Ca(2+) receptor (CaR) possesses an approximately 600-residue extracellular domain involved in ligand binding and receptor activation. Based on an alignment of the amino acid sequence of the CaR with that of bacterial periplasmic-binding proteins, the first approximately 530 residues of the extracellular domain are believed to form a domain resembling a bilobed Venus's flytrap (VFT). Four insertions in the CaR sequence that do not align with those of bacterial periplasmic-binding proteins correspond to four loops within lobe I of the VFT. We constructed a series of deletion mutants of these four loops and tested their ability to form fully processed CaR as well as their ability to be activated by Ca(2+). As many as 21 residues (365) of loop III could be deleted without impairing receptor expression or activation. Deletion of portions of either loops I (50) or IV (438) did not impair receptor expression but significantly reduced Ca(2+) activation. Deletion of the entire loop II (117) abolished receptor expression and function, but the replacement of even a single residue within this deletion mutant led to expression of a monomeric form of the receptor showing increased Ca(2+) sensitivity but reduced maximal activation. Our results reveal that certain residues within loops I and IV are dispensable in formation of the VFT domain but are critical for Ca(2+) activation of the receptor. In contrast, the residues in loop II are critical for maintaining the inactive state of the CaR. We discuss these results in light of the recently defined crystal structure of the homologous domain of the type 1 metabotropic glutamate receptor.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Mutagénesis Sitio-Dirigida , Eliminación de Secuencia , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Línea Celular , Humanos , Datos de Secuencia MolecularRESUMEN
The extracellular N-terminal domain of the human Ca(2+) receptor (hCaR) consists of a Venus's-flytrap (VFT) domain and a cysteine-rich (Cys-rich) domain. We have shown earlier that the Cys-rich domain is critical for signal transmission from the VFT domain to the seven-transmembrane domain. The VFT domain contains 10 cysteines: two of them (Cys(129) and Cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (Cys(60)-Cys(101), Cys(358)-Cys(395), and Cys(437)-Cys(449)) are predicted to form three intramolecular disulfide bonds. The Cys-rich domain contains nine cysteines, the involvement of which in disulfide bond formation has not been defined. In this work, we asked whether the remaining cysteines in the hCaR VFT, namely Cys(236) and Cys(482), form disulfide bond(s) with cysteines in the Cys-rich domain. We constructed mutant hCaRs with a unique tobacco etch virus (TEV) protease recognition site inserted between the VFT domain and the Cys-rich domain. These mutant hCaRs remain fully functional compared with the wild type hCaR. After TEV protease digestion of the mutant hCaR proteins, dimers of the VFT were identified on Western blot under nonreducing conditions. We concluded that there is no disulfide bond between the VFT and the Cys-rich domains in the hCaR.
Asunto(s)
Proteínas de Unión al Calcio/química , Cisteína/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Dimerización , Disulfuros , Relación Dosis-Respuesta a Droga , Endopeptidasas/metabolismo , Ácido Glutámico/química , Humanos , Immunoblotting , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Transfección , Valina/químicaRESUMEN
The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/L (Des-Arg(9))-Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Receptores de Bradiquinina/genética , Spodoptera/metabolismo , Animales , Baculoviridae/genética , Bradiquinina/farmacología , Antagonistas de los Receptores de Bradiquinina , Calcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Unión al GTP/metabolismo , Expresión Génica , Genisteína/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Microscopía Confocal , Octopamina/farmacología , Ácidos Fosfoaminos/análisis , Fosforilación , Unión Proteica , Receptor de Bradiquinina B2 , Receptores de Bradiquinina/agonistas , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/metabolismo , Tapsigargina/farmacología , Tirfostinos/farmacologíaRESUMEN
The prevalence of beta thalassemia (B-thal) in Mexico is largely unknown, and it is thought that the disease is confined to populations with Mediterranean ancestors. Various reports suggest that in certain parts of the coast in the Gulf of Mexico the prevalence of both B-thal and hemoglobin S disease/trait is high. We studied prospectively a town with 11,000 inhabitants named Tamiahua, located along the Gulf Coast, in the State of Veracruz, and very close to the State of Tamaulipas. A group of 200 inhabitants was initially studied: the prevalence of B-thal was 15% and 6% of them had sickle cell trait. The prevalence of B-thal is the highest reported in the country. In a second part of the study, two family trees with members heterozygous for B-thal and/or Hb S trait were constructed. The ethnic characteristics of the studied population makes unlikely that the gene was derived from white Europeans but not from black Africans. Inasmuch as the indians living in that part of the country belong to the macro-maya glotochronological group, where a relatively high prevalence of B-thal has also been identified, we feel that it is possible that B-thal was present in our country before the Spaniards arrived to Mexico.