Critical Role of Electrostatic Interactions of Amino Acids at the Cytoplasmic Region of Helices 3 and 6 in Rhodopsin Conformational Properties and Activation

Abstract

The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cisretinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr 251 in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.

Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

“…Mutations of 6 . 30 cause constitutive activation in rhodopsin (Ramon et al 2007), b 2 -adrenoceptor (Ballesteros et al 2001), and lutropin receptor (Angelova et al 2002). The salt bridge was indeed observed in the crystal structure of rhodopsin (Palczewski et al 2000).…”

The 1D4 Antibody Labels Outer Segments of Long Double Cone But Not Rod Photoreceptors in Zebrafish

“…9 A peptide comprising the C-terminal eight amino acids E-T-S-Q-V-A-P-A was used to map the binding sites of 1D4. 10,11 Given its high specificity, it has been widely used in studies addressing structure and function of rhodopsin, 12 as well as a reliable fusion tag for purification of nonrelated receptors. 13,14 In mice, it has been used for the detection of rhodopsin 15 and its subcellular distribution 16 during retinal degeneration.…”

Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

“…This includes also interactions associated to the “ionic lock” located at the cytoplasmic ends of the third and sixth transmembrane helices, respectively, (positions: 3.49, 3.50, 3.51, 6.30, and 6.34 in the GPCR family) play an important role in the receptor activation mechanism [50, 63]. …”

“…Among the residues listed in Figure 7, those forming the so-called ionic-lock between TM3 and TM6, in subscript the Ballesteros-Weinstein numbering is used [49]: D165 3.49 , R166 3.50 , Y167 3.51 and E486 6.30 , R253 5.60 and R180 4.37 are indicated. Electrostatic interactions at these positions are known to be critical for restraining the receptor from adopting a constitutively active GPCR [50, 51]. Some of the interactions are only found in the two POPS/POPC simulations: D142-K213, D165-R166, E259-R480, E220-Y128, E220-Y530, and N548-T50.…”