Comparison of Class A and D G Protein-Coupled Receptors:  Common Features in Structure and Activation

Abstract

All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure-function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast α-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors.G protein-coupled receptors (GPCRs) 1 are the largest and most diverse superfamily of membrane receptors. GPCRs are characterized by their common 7 transmembrane (TM) helix architecture and their ability to activate heterotrimeric G proteins. Surprisingly, there is no significant sequence similarity across the GPCR superfamily. In fact, GPCRs have been grouped into at least five distinct classes (1) with no recognizable sequence similarity between the various classes (1,2). Nevertheless, the working assumption has been that these receptors have a common fold and activation mechanism. For example, the amino acid sequences of the yeast pheromone GPCRs, such as the α-factor receptor, diverge considerably from those of the mammalian hormone GPCRs, such as the β-adrenergic receptor. Yet, in both of these receptors, ligand binding occurs within the core of the 7 TM helices and is, in part, mediated by aromatic amino acids on transmembrane helix H6

The KlSTE2 and KlSTE3 genes encode MATα‐ and MATa‐specific G‐protein‐coupled receptors, respectively, which are required for mating of Kluyveromyces lactis haploid cells

“…When comparing K. lactis receptors with their S. cerevisiae homologues, most conservation occurs in residues that nucleate transmembrane helices (Figure 1). Mostly, key amino acids that have been involved in interhelical contacts, in resting and active states of the receptor, are preserved in both proteins (Eilers et al, 2005). Regarding the extra-membrane segments, the region most highly conserved is the third cytoplasmic loop in both proteins, 17 out of 21 for the Ste2p receptors, and 31 out of 36 for the Ste3p receptors (Figure 1).…”

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

“…Ste2 activates a heterotrimeric G protein in which the α subunit shows about 45% identity with mammalian Gα proteins and is most closely related to the Gi subfamily (4). In addition, comparison of Ste2 with rhodopsin, a member of the large class A subfamily of mammalian GPCRs, indicates that there are similar microdomains in these divergent receptors (5). These results suggest that there are underlying similarities in the mechanisms of signal transduction by the diverse GPCR family.…”

“…The conserved residues were identified by aligning 28 Ste2 sequences from a broad range of yeast and filamentous fungi (5). The majority of the conserved residues are in the intracellular half of Ste2 (Fig.…”

“…The colored bars represent the ends of the TMDs predicted by the TMHMM and SOSUI computer algorithms (40) that were accessed at the ExPASy molecular biology server (http://ca.expasy.org/). (B) Circles identify residues with the indicated percent identity across 28 different members of the Ste2 family of fungal pheromone receptors (5). Subfamily conserved residues show a high degree of identify within the budding yeast and filamentous fungi subfamilies, but are not identical between the two subfamilies.…”

“…Cys substitution mutations were introduced into the residues that span across the intracellular loops and the cytoplasmic end of TMD7 so that their solvent accessibility could be probed with MTSEA-biotin, a thiol reactive probe that does not react with membrane-buried residues (12). A similar approach used to define that topology of the extracellular regions of Ste2 demonstrated that the extracellular ends of the TMDs play a key role in ligand binding and in promoting the active receptor conformation (13,14), and also provided a structural framework for comparing Ste2 to mammalian GPCRs (5). Therefore, the accessibility of residues in the cytoplasmic regions of Ste2 was assayed to provide a structural framework for understanding the role of residues that function in G protein activation.…”