Most of the current knowledge about structure/function relationships of GPCRs is based on studies with rhodopsin and other members of the Family 1 of GPCRs and the mechanics of receptor activation are covered in more detail in Chapter 3. Only a few critical amino acids have been preserved during evolution of the rhodopsin-like GPCR family (Fig. 1.3). Despite an evolutionary conservation mutational alteration of some conserved amino acid residues does not always have the same functional consequence.
The majority of Family 1 and also Family 2 GPCRs contains a conserved pair of extracellular cysteine residues linking the first and second extracellular loops via a disulfide bond. Numerous functional analyses of mutant GPCRs in which the cysteine residues were replaced by other amino acids have shown that this disulfide bond maybe critical for receptor signalling (Wess 1997). But in some receptors this disulfide bond is required to maintain more distinct functions. Systematic mutagenesis studies of the conserved cysteine residues in several GPCRs showed that disruption of the disulfide bond does not influence the receptor's ability to activate G protein, but interferes with high affinity ligand binding and receptor trafficking (Le Gouill etal. 1997; Perlmann etal. 1995; Schulz etal. 2000a; Zeng etal. 1999). Although the disruption of the disulfide bond, the receptor core structure appears to remain intact, allowing receptor function. Consistent with this notion, some GPCRs, for example, receptors for sphingosine 1-phosphate and lysophosphatidic acid, lack the conserved extracellular
Cys residues. Interestingly, many GPCRs including receptors for biogenic amines, peptides and many 'orphan' GPCRs contain a second conserved pair of extracellular cysteine residues linking the N terminus and third extracellular loop (see Fig. 1.3b). Mutational disruption of this disulfide bond results in a loss of high affinity binding of receptor ligands, suggesting a pivotal role of an N terminus/e3 loop-connecting disulfide bridge for proper receptor assembly (Ho et al. 1999; Hoffmann et al. 1999). In the crystal structure of rhodopsin, the N-terminal segment is located just below the e3 loop. Specific non-covalent contacts maintain the proper orientation between the rhodopsin N terminus and the extracellular loops so that an additional disulfide bridge like in other GPCRs is probably not required.
Most Family 1 GPCRs possess within their transmembrane core a number of highly conserved residues, such as an Asp residue in TMD2, a DRY motif at the TMD3/i2-transition, a Trp residue in TMD4, a Tyr residue in TMD5, a Pro residue in TMD6 and an N/DPXXY motif in TMD7 (Fig. 1.3a). For example, the DRY motif located at the boundary of TMD3 and the i2 loop is a highly conserved triplet of amino acid residues known to play an essential role in GPCR function (see Chapter 3). The crystal structure of rhodopsin proposes that the residues of the DRY motif participate in several hydrogen bonds with surrounding residues of TMD6. However, the fine structure of this region has not been finally resolved (Palczewski et al. 2000). It is also of note that several Family 1 GPCRs are known in which the acidic residue (Asp, Glu) within this motif is naturally substituted by His, Asn, Gln, Gly, Val, Thr, Cys, or Ser residues, questioning the general importance of a protonation event at this amino acid position for GPCR function. The fully conserved Arg residue in the DRY motive is considered to be a key residue in signal transduction of GPCRs. Replacement of the conserved Arg residue by different amino acids virtually abolished G protein coupling of many GPCR (Franke etal. 1992; Jones etal. 1995; Scheer etal. 1996). Therefore, the conserved Arg residue has been implicated as a central trigger of GDP release from the G protein a subunit (Acharya and Karnik 1996). Recent studies with mutants of the N-formyl peptide receptor, LHR and V2 vasopressin receptor (AVPR2) showed that, G protein coupling is only decreased, but not abolished after replacement of the Arg residue in the DRY motif (Arora et al. 1997; Schoneberg et al. 1998; Schulz et al. 1999) probably due to reduced receptor cell surface expression levels as response to constitutive arrestin-mediated desensitization (Barak etal. 2001).
The N/DPXXY motif at the cytoplasmic end of TMD7 is one of the most conserved regions among the Family 1 GPCRs (Fig. 1.3). It was shown that light activation of rhodopsin made an epitope including residues of the N/DPXXY motif accessible for an epitope-specific antibody, suggesting conformational changes of this sequence motif (Abdulaev and Ridge 1998). The Asn residue in this sequence motif is thought to play a crucial role in receptor activation and signal transduction specificity by involving small G proteins such as ARF and RhoA (Mitchell et al. 1998). In the cholecystokinin type B receptor, mutation of the Asn in the NPXXY motif to Ala had no effect on cell surface expression and high affinity ligand binding, but completely abolished Gq-mediated signalling (Gales et al. 2000). Mutational alteration of the conserved Pro residue in the N/DPXXY motif resulted in a complete loss of receptor function as demonstrated in vivo for the AVPR2 (Tajima et al. 1996) underlining the functional importance of this motif.
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