Herramientas de la etapa de ajuste y selección

The transmembrane regions comprise o f 20-25 hydrophobic amino acids postulated to form a-helical domains. The seven transmembrane domains (TMDs) form a narrow

Introduction D R D l V D R D 5 - 1 VDRD5-2 DRD5 D RD3 D R D 2 D R D 4 Figure 1.7: P h y lo g en etics o f the d o p am in e receptors, based on the rat and the h um an genes (adapted from V aldenaire et a l., 1994 and M archese a /., 1995). y -p s e u d o g e n e s D RDl DRD5 DRD2 V //A V /A V //A VM W A I III IV VI VII I II III IV -w r v /A III IV m x r jy m V VI VII m vm m VI vu DRD3 I I n ni IV V TVH l | i f DRD4 _{m m m} I II -ECm-III IV k kL Ua a a VI VII S r - m m VI vu

Figure 1.8; Genomic organisation of the five dopam ine receptor genes (adapted from

Civelli et a l, 1993). Exons are indicated by the boxes and introns are indicated by the horizontal lines

Location of the seven putative transm em brane dom ains U ntranslated regions of the corresponding mRNA illLil Alternatively spliced exon differentiating D2A and D2B I'"''') Seven 48bp repeats of the DRD4

Introduction dihedral hydrophobic cleft surrounded by alternating extracellular and intracellular loops, composed o f hydrophilic residues (see figure 1.9). Molecular models indicate that the charged residues o f these a-helices that interact with dopamine face the inside part o f the hydrophobic cleft. These are: two aspartate residues in the second and third TM D , which form an ion pair with the protonated amine group o f dopamine (see figure 1.9 and figure 1.10), with the carboxylate side-chains serving as counterions to the protonated amine groups; two or three serine (Ser) residues in the fifth TMD form a hydrogen bond with the two phenol groups o f dopamine, this interaction, which is specific for dopamine and its agonists, is thought to cause a conformational change in the helix that is transmitted to the third cytoplasmic loop; two cysteine (Cys) residues between amino acid position 103-108 and 178-185 o f the extracellular loop 1 and 2 respectively, which are envisaged to serve a stabilising factor on the tertiary structure o f the receptor by forming a disulphide bridge (see figure 1.11). See table 1.2 for the amino acids o f the five dopamine receptors involved in the interaction with dopamine.

The receptor polypeptides are anchored to the membranes through palmitoylation (acylation with palmitic acid) o f a conserved cysteine residue in the C-terminus (see figure 1.11 and 1.12, table 1.2). This type o f covalent modification is thought to anchor the C-terminal tail o f the receptor to the internal face o f the plasma membrane, thereby creating a fourth cytoplasmic loop. This fourth cytoplasmic loop has been suggested to take part in the G-protein coupling event. The N-terminal stretch o f all dopamine receptors has a similar number o f amino acids and carries 1-6 putative N-linked glycosylation sites at asparagine (Asn) residues (see figure 1.11 and 1.12). Glycosylation probably has a structural role (see table 1.2 for the a.a glycosylated in the different receptors).

The third cytoplasmic domain is rich in serine and threonine residues, some o f which are phosphorylated by cAMP-dependent protein kinase or protein kinase C (see figure 1.11), and exhibits the largest sequence dissimilarity among the various dopamine receptor subtypes. This region has been suggested to provide selective interaction o f each subtype with one member o f the large family o f G-proteins, leading to distinct intracellular signals. Biochemical studies have shown that phosphorylation o f a number o f the serine and threonine residues may attenuate G protein-receptor interactions and that these residues are directly involved in the homologous and heterologous mechanisms

Extracellular space E x tra c e llu la r / loops In tracellu lar loops L ip id b ilay er C vtosol

F igu re 1.9: A sch em atic rep resen tatio n o f the cross sectio n o f the lipid b ilay er sh o w in g the interaction o f the d o p am in e m o lecu le (green) w ith a m n io acids o f the tran sm em b ran e d o m ain s o f the D R D 4 (red, ad ap ted from T au b es, 1994).

C168

C56

S 1 9 3 8 1 9 4 ^ 0 C126 D80

VII

F igu re 1.10: R elativ e p o sitio n s o f the seven tran sm em b ran e helices in the m id -p lan e o f the m em b ran e and their o rien tatio n to w a rd s the d o p am in e b in d in g -site crev ice (ad ap ted from Ja v itc h e t al., 1994).

Human DRDl Human DRD2 V I I Human DRD5 Human DRD3 V I I W V I I Human DRD4 [= □ Y s-s - Plasma membrane - Transmembrane domain

- The extracellular and intracellular loops - N-linked glycosylation sites

- The two cysteine residues involved in forming the disulfide bridge

- Palmitoylated cysteine residue

- Sites phosphorylated or dephosphorylated by cAMP dependent protein kinase - Sites phosphorylated by protein kinase C

.s-s

V I I

Figure 1.11: Comparison of the structural features of the five dopamine receptors, a schematic representation (adapted from Gingrich and Caron, 1993).

The Human 02^43 R eceptor