Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β(2)-adrenergic receptor

Abstract

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix-helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2-H4 in the β(2)-adrenergic receptor (β(2)-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315(7.42) and Ser319(7.46), on H7 for structure-function analysis. Replacing Ser319(7.46) with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15-20% agonist-independent activity. Replacement of Ser319(7.46) with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315(7.42) and Ser319(7.46) are stabilizing β(2)-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315(7.42) with Trp286(6.48) and hydrogen bonding interactions of Ser319(7.46) with amino acids on H1-H2-H7 and with structural water.

Figure 1

Two-dimensional representation of the β₂-AR sequence showing amino acid conservation at the three different levels. The receptor has seven transmembrane helices (H1–H7) and a short amphipathic helix (H8) that lies on the cytoplasmic surface of the membrane. Amino acids are shown in single-letter codes. Red circles denote signature-conserved residues that include the (E/D)RY and NPxxY motifs, an asparagine (Asn51^1.50) on H1, aspartic acid on H2 (Asp79^2.50), three prolines on helices H5 (Pro211^5.50), H6 (Pro288^6.50) and H7 (Pro323^7.50) and several hydrophobic residues (Leu75^2.46,Trp158^4.50). Blue circles represent small and weakly polar group–conserved residues in the Class A GPCRs excluding the olfactory subfamily, and green circles subfamily-specific residues. The residues in gray are between 70% and 90% conserved in the visual receptor subfamily. CL, cytoplasmic loop; CT, C terminus; EL, extracellular loop; NT, N terminus.

Figure 2

Immunofluorescence microscopy showing localization of wild-type β₂-AR and the group-conserved mutants expressed in HEK293T cells. Immunofluorescence was performed with the mouse rho-1D4 antibody (A) and the endoplasmic reticulum marker, rabbit anti-calnexin antibody (B). The mouse rho-1D4 antibody was visualized with anti-mouse-FITC secondary antibody (green), and the rabbit anti-calnexin antibody was visualized with anti-rabbit-Texas Red secondary antibody (red). Overlays of (A) and (B) are shown in (C). Yellow arrows show the locations of receptors. In the wild-type, S319A, S319G and G315A mutants, the receptors were predominantly localized at the plasma membrane, whereas in G315S, G315L and S319L the receptors appear to be located in intracellular compartments.

Figure 3

Characterization of Gαs-mediated signaling of the wild-type and group-conserved helix H7 mutants of β₂-AR. Receptor activity was determined by measuring cAMP production using transiently transfected HEK293S cells. Shown are the basal (zero concentration) and agonist (isoproterenol) induced cAMP production of the wild-type receptor and the Gly315 and Ser319 mutants. Arrows indicate an increase in the basal activity of the mutants over the wild type receptor. The results are expressed as a percentage of the wild type β₂-AR activity, and are from at least three independent experiments performed in duplicate.

Figure 4

Molecular packing of Gly315^7.42 and Leu315^7.42 in β₂-AR. (A) Crystal structure of β₂-AR (2RH1) showing van der Waals interaction of Trp286^6.48 and Gly315^7.42. (B) Homology model of G315L β₂-AR where the G315L β₂-AR sequence was threaded onto the structure of active opsin (3CAP) and the resulting structure was energy minimized with 1000 steps steepest descent. The inactive structure of rhodopsin (C) and the active structure of opsin (D) are shown for comparison.

Figure 5

Crystal structure of the β₂-AR in the region of Gly315^7.42 and Ser319^7.46. (A) Packing of Gly315^7.42 with Trp286^6.48. An increase in size of the amino acid at position 7.42 causes a steric clash with Trp286^6.48 on H6 and is predicted to displace the Trp286^6.48 side chain toward the extracellular surface. (B) Hydrogen bonding interactions of Ser319^7.46. The side chain hydroxyl group of Ser319^7.46 side chain is hydrogen bonded to the carboxyl C(O)OH of Asp79^2.50 (3.3 Å), to the backbone carbonyl of Tyr316^7.43 (3.1 Å) and to water 534 (3.1 Å), while the backbone carbonyl of Ser319^7.46 makes contact with the side chain NH of Asn51^1.50 (3.1 Å). Water 534 is shown hydrogen bonded to the backbone of Gly315^7.42. (C) Hydrogen bonding network connecting the indole NH of Trp286^6.48 to the amide side chain of Asn322^7.49. Water 534 in β₂-AR mediates hydrogen bonding between the Trp286^6.48 indole NH (3.48 Å), Ser319^7.46 CβOH (2.82 Å), Asn318^7.45 NH₂ (3.11 Å) and Gly315^7.42 (C=O) (3.38 Å). Water 534 is also hydrogen bonded to Wat548, which in turn is hydrogen bonded to Asn322 through Wat532. The same overall set of interactions appears to exist in the recent crystal structure of the CXCR4 receptor where cysteine occurs at position 7.46 and histidine occurs at position 7.45 [24]. The polar imidizole of histidine is located between the conserved indole ring of Trp6.48 and Cys7.46.