Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase
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S B Masters1, R T Miller, M H Chi
1Department of Pharmacology, University of California, San Francisco 94143.
Abstract:
Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa ras proteins (p21ras) reduce their GTPase activity. To test the possibility that the cognate regions of G protein alpha chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated alpha chains (termed alpha s) of Gs, the stimulatory regulator of adenylyl cyclase. alpha s chains were expressed in an alpha s-deficient S49 mouse lymphoma cell line, cyc-. alpha s in which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21ras) constitutively activates adenylyl cyclase and reduces the kcat for GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21ras). This mutant alpha s is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal alpha s, is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that alpha s residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of alpha s and p21ras are not identical.
Insights
Mutations in G protein alpha s (alpha s) chains impact GTP binding and hydrolysis, affecting adenylyl cyclase activity. These findings reveal key residues involved in G protein signaling and GTPase function.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Signaling
Background:
- Ras proteins (p21ras) have GTP-binding pockets that regulate GTPase activity.
- G protein alpha subunits (alpha s) are crucial regulators of adenylyl cyclase.
- Understanding alpha s function is key to deciphering cellular signaling pathways.
Purpose of the Study:
- To investigate if G protein alpha s chains participate in GTP binding and hydrolysis.
- To compare the signaling functions of normal and mutated alpha s chains.
- To elucidate the role of specific residues in alpha s GTPase activity.
Main Methods:
- Expression of normal and mutated alpha s chains in an alpha s-deficient cell line (cyc-).
- Assay of adenylyl cyclase activity in response to alpha s mutations.
- Measurement of GTP hydrolysis rates (kcat) for wild-type and mutant alpha s proteins.
- Assessment of protection against tryptic cleavage by GTP analogs.
Main Results:
- A mutation at glutamine 227 (leucine substitution) constitutively activated adenylyl cyclase and reduced GTP hydrolysis by over 100-fold.
- A mutation at glycine 49 (valine substitution) resulted in poor adenylyl cyclase activation and reduced GTPase activity.
- The glycine 49 mutant showed impaired ability to attain the active GTP-bound conformation, as indicated by lack of protection from tryptic cleavage.
Conclusions:
- Specific residues in alpha s, particularly near glutamine 227 and glycine 49, are critical for GTP binding and hydrolysis.
- The GTP-binding regions of alpha s and p21ras share functional similarities but are not identical.
- These findings highlight the conserved yet distinct mechanisms of GTP regulation in different GTPase families.