Receptor and Membrane Interaction Sites on Gβ

Abstract

The functional organization of Gbetagamma is poorly understood. Regions of bovine brain Gbetagamma that interact with a photoaffinity derivative of an alpha2-adrenergic receptor-derived peptide from the third intracellular loop (diazopyruvoyl-modified peptide Q (DAP-Q)) and a hydrophobic membrane probe (3-trifluoromethyl-3-(m-iodophenyl)diazirine (TID)) were examined. We previously showed that DAP-Q cross-links to specific, competable sites on both the alpha and beta subunits of Go/Gi but not on the gamma subunit and that betagamma subunit was required for stimulation of Go/Gi GTPase activity (Taylor, J. M., Jacob Mosier, G. G., Lawton, R. G., Remmers, A. E., and Neubig, R. R. (1994) J. Biol. Chem. 269, 27618-27624). Similarly, we show here that the membrane-associated photoprobe [125I]TID labels alpha and beta but not gamma. We have now mapped the sites of incorporation of DAP-Q and TID into the beta subunit. TID labels both the 14-kDa amino-terminal and the 23-kDa carboxyl-terminal fragments from a partial tryptic digest of beta while DAP-Q labels only the carboxyl-terminal fragment. Further mapping with endopeptidase Lys C reveals substantial labeling of multiple fragments by TID while DAP-Q labels predominantly a approximately 6-kDa fragment within the carboxyl-terminal 60 amino acids of beta1. Thus, regions within the 7th (or possibly 6th) WD-40 repeat of the beta subunit of G protein interact with the receptor-derived peptide while membrane interaction involves multiple sites throughout the beta subunit.

Rescue of Functional Interactions between the α2A-Adrenoreceptor and Acylation-resistant Forms of Gi1α by Expressing the Proteins from Chimeric Open Reading Frames

“…These results imply interaction of the fusion protein with the ␤␥ complex, but understanding the details of this will require further study. It is of interest in this regard to note, however, that Taylor et al (36) have previously indicated a role for ␤␥ in receptor stimulation of the GTPase activity of the ␣ 2A -adrenoreceptor.…”

“…Given the nature of the physical linkage between the receptor and G protein in the fusion proteins, the knowledge that the N terminus of the G protein ␣ subunit plays a central role in interaction with the ␤␥ complex (28 -29), the concept that the ␤␥ complex may play a key role in receptor interactions with the ␣ subunit (33)(34)(35), and the understanding that G protein ␣ subunit acylation is important in interactions with the ␤␥ complex, it was of considerable interest to observe that coexpression of excess ␤ 1 ␥ 2 along with any of the ␣ 2A -adrenoreceptor-G i1 ␣ fusion proteins resulted in greater maximal UK14304 stimulation of GTPase activity (Fig. 5).…”

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β‐adrenoceptor

“…This portion together with the flexible part could serve as a counterpart to the bipartite receptor recognition site on the G protein. specifically be cross-linked to both the ct-and fl-subunits of the corresponding G protein [31]. Mastoparan, on the contrary, can form only cross-links to the ~subunit [32], indicating that mastoparan is able to fulfill only one aspect of the structural requirements for G protein coupling.…”

A Cyclic Peptide Mimicking the Third Intracellular Loop of the V2 Vasopressin Receptor Inhibits Signaling through Its Interaction with Receptor Dimer and G Protein

“…For the rat vasopressin V 1a receptor, a peptide mimicking its second intracellular loop was found to specifically inhibit vasopressin-specific binding (2). In other cases, short peptides mimicking GPCR third intracellular loops were found to act on the coupling between the receptor and the G protein (3)(4)(5)(6)(7)(8). Expressed as a minigene, the ␣ 1b -adrenergic i3 loop was shown to inhibit Gq signaling (9).…”