Identification of a stretch of six divergent amino acids on the alpha5 helix of Galpha16 as a major determinant of the promiscuity and efficiency of receptor coupling

Abstract

A broad repertory of G-protein-coupled receptors shows effective coupling with the haematopoietic G16 protein. In the present study, individual residues along the C-terminal alpha5 helix of Galpha16 were examined for their contributions in defining receptor-coupling specificity. Residues that are relatively conserved within, but diverse between, the subfamilies of cloned Galpha subunits were mutated into the corresponding Galpha(z) residues. Six G(i)-linked receptors with different coupling efficiencies to Galpha16 were examined for their ability to utilize the various Galpha16 mutants to mediate agonist-induced inositol phosphate accumulation and Ca2+ mobilization. Co-operative enhancements of receptor coupling were observed with chimaeras harbouring multiple mutations at Glu350, Lys357 and Leu364 of Galpha16. Mutation of Leu364 into isoleucine appeared to be more efficient in enhancing receptor recognition compared with mutations at the other two sites. Mutation of a stretch of six consecutive residues (362-367) lying towards the end of the alpha5 helix was found to broaden significantly the receptor-coupling profile of Galpha16, and the effect was mediated partly through interactions with the beta2-beta3 loop. These results suggested that a stretch of six distinctive residues at the alpha5 helix of Galpha16 is particularly important, whereas other discrete residues spreading along the alpha5 helix function co-operatively for determining the specificity of receptor recognition.

A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

“…Biochemical, kinetic, and structural characterizations have identified binding sites for GPCR on the G␣ subunit (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). This contiguous surface includes the N terminus (2-7), the ␣4-␤6 loop (8 -13), the ␣3-␤5 loop (14), and the C terminus of G␣ (3) and is located Ͼ30 Å away from the guanine nucleotide binding site (15)(16)(17) (Fig.…”

G Proteins in Drug Screening: From Analysis of Receptor-G Protein Specificity to Manipulation of GPCR-Mediated Signalling Pathways

“…(v) The serotonin 5-HT1B receptor couples to G protein heterotrimers containing either Gαi1, Gαi2, Gαi3, or Gαo but not Gαt although their Ctermini are highly homologous [71]. To date, at least 4 other regions within Gα have been shown to be involved in receptor interaction: The amino-terminal domain [21,[72][73][74], the α2 helix and α2-β4 loop regions [26,27], the α4 helix and α4-β6 loop domain [26,28,61,75,76], the α5 helix [77,78], the α3-β5 region [79], and a small segment within the loop linking the N-terminal α helix to the β1 strand of the ras-like domain [32]. Of those, the extreme N-and Ctermini, the αN-β1 loop, the α4-β6 region, and the α5 helix contribute to the specificity of Gα-receptor interaction ( Fig.…”

“…Interestingly, most of the GPCRs that fail to interact with Gα16 are Gi-selective [for a comprehensive list see refs in 76,98,99]. Therefore attempts have been made to increase the promiscuity of Gα16 towards the Gi family of receptors [76,78,99]. Mody et al [76] created a series of chimeras between Gα16 and Gαz and identified two chimeric Gα proteins to be particularly useful: Gα16z25 and Gα16z44 in which the sequence of the α4-β6 and α5 regions were derived from Gαz.…”

A Hematopoietic Perspective on the Promiscuity and Specificity of Gα₁₆ Signaling

“…Further efforts have been made to decipher the minimal requirements for switching the receptor-coupling specificity. Individual residue lying on the exposed surface of the ␣ 5 helix of G ␣ 16 , as well as a stretch of six amino acids between the ␣ 5 helix and the extreme C-terminal tail, plays an important role in determining the receptor-coupling specificity [30] . Other potential receptor-interacting regions on G ␣ 16 have also been explored, such as the ␤ 2/ ␤ 3 loop [30] , ␣ N/ ␤ 1 loop [31] , as well as the uniquely long ␣ 4/ ␤ 6 loop ( fig.…”

“…The same strategy worked equally well for G s -coupled receptors [27] . Chimeric G ␣ 16 subunits with broadened receptor-coupling capability have been successfully employed in high throughput screening platforms [23,[28][29][30] . Further efforts have been made to decipher the minimal requirements for switching the receptor-coupling specificity.…”