A high temperature transfer procedure for detection of G protein gamma subunits by immunoblotting.

The production of antibodies against synthetic peptides derived from amino acid sequences common or unique to a particular protein(s) is an important tool in the identification of structurally related members of an ever-increasing number of protein families. The successful use of anti-peptide antibodies requires that the protein(s) of interest be properly transferred and fully denatured prior to detection by Western-type immunoblotting. In this paper, we demonstrate that conventional transfer procedures are not successful in presenting the G protein gamma subunits in a suitable state for immunodetection. We describe a high temperature (70 degrees C) transfer procedure that results in a more than 20-fold enhancement in the sensitivity of immunodetection of the various G protein gamma subunits. The effect of high temperature transfer could not be duplicated by including 0.2% SDS in the buffer during transfer to nitrocellulose, or by baking or autoclaving the nitrocellulose after transfer. Thus, high temperature transfer is a powerful procedure for enhancing immunoblot detection of protein(s) that may be resistant to denaturation and/or subject to renaturation during the transfer and/or binding to nitrocellulose.

Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning.

The GTP-binding regulatory proteins (G proteins) that transduce signals from receptors to effectors are composed of alpha, beta, and gamma subunits. Whereas the role of alpha subunits in directly regulating effector activity is widely accepted, it has recently been demonstrated that beta gamma subunits may also directly regulate effector activity. This has made clear the importance of identifying and characterizing beta and gamma subunits. We have isolated a cDNA clone encoding a new gamma subunit, referred to here as the gamma 7 subunit, using probes based on peptide sequences of a gamma subunit previously purified from bovine brain. The clone contains a 1.47-kilobase cDNA insert, which includes an open reading frame of 204 base pairs that predicts a 68-amino acid polypeptide with a calculated M(r) of 7553. The predicted protein shares amino acid identities with the other known gamma subunits, ranging from 38 to 68%. Also characteristic of gamma subunits is a carboxyl-terminal CAAX motif. The expression of the gamma 7 subunit as well as the gamma 2, gamma 3, and gamma 5 subunits was examined in several bovine tissues at both the mRNA and protein levels. Whereas the gamma 2 and gamma 3 subunits were selectively expressed in brain, the gamma 5 and gamma 7 subunits were expressed in a variety of tissues. Thus, the gamma 5 and gamma 7 subunits are the first G protein gamma subunits known that could participate in the regulation of widely distributed signal transduction pathways.

Human G protein gamma(11) and gamma(14) subtypes define a new functional subclass.

The mammalian gamma subunit family consists of a minimum of 12 members. Analysis of the amino acid sequence conservation suggests that the gamma subunit family can be divided into three distinct subclasses. The division of the gamma subunit family into these classes is based not only on amino acid homology, but also to some extent on functional similarities. In the present study, two new members of the gamma subunit family, the gamma(11) and gamma(14) subunits, are identified and characterized in terms of their expression and function. The gamma(11) and gamma(14) subunits are most closely related to the gamma(1) subunit and share similar biochemical properties, suggesting their inclusion in class I. However, despite their close phylogenetic relationship and similar biochemical properties, the gamma(1), gamma(11), and gamma(14) subunits exhibit very distinct expression patterns, suggesting that class I should be further subdivided and that the signaling functions of each subgroup are distinct. In this regard, the gamma(11) and gamma(14) subunits represent a new subgroup of farnesylated gamma subunits that are expressed outside the retina and have functions other than phototransduction.

Bovine brain GO isoforms have distinct gamma subunit compositions.

The gamma subunit composition of the major bovine brain Go and Gi proteins (GOA, GOB, GOC, Gi1, and Gi2) was characterized using antibodies against specific gamma isoforms. Each of the purified G protein heterotrimers contained a heterogeneous population of gamma subunits, and the profiles of the gamma subunits found with Gi1, Gi2, and GOA were similar. In contrast, each GO isoform had a distinct pattern of associated gamma subunits. These differences were surprising given that all three alpha O isoforms are thought to share a common amino-terminal sequence important for the binding of beta gamma dimers and that the alpha OA and alpha OC proteins may come from the same alpha O1 mRNA. The free alpha OA and alpha OC subunits had unique elution behaviors during MonoQ chromatography, compatible with differences in their post-translational processing. These results indicate that both the alpha and gamma subunit compositions of heterotrimers define the structure of an intact G protein. Furthermore, the exact subunit composition of G protein heterotrimers may depend upon regulated expression of different subunit isoforms or upon cellular processing of alpha subunits.