Rhodopsin-family receptors associate with small G proteins to activate phospholipase D

Area of Science:

• Molecular biology
• Cell signaling
• Biochemistry

Background:

• G-protein-coupled receptors (GPCRs) are crucial for neural and endocrine signaling.
• GPCRs activate heterotrimeric G proteins and can also modulate phospholipase D (PLD) activity.
• PLD can be regulated by small G proteins, including ARF and RhoA.

Purpose of the Study:

• To investigate the role of small G proteins ARF and RhoA in GPCR-mediated phospholipase D activation.
• To identify structural determinants within GPCRs that govern their interaction with ARF and RhoA.
• To elucidate the mechanism by which specific GPCRs activate PLD.

Main Methods:

• Co-immunoprecipitation assays to detect receptor-G protein interactions.
• GTP analog binding assays to assess receptor-G protein coupling.
• Site-directed mutagenesis to alter specific amino acid sequences within GPCRs.
• Pharmacological inhibition studies using ARF and RhoA inhibitors.

Main Results:

• Many Ca2+-mobilizing GPCRs activate phospholipase D in an ARF- and RhoA-dependent manner.
• GPCRs that interact with ARF/RhoA possess an AsnProXXTyr motif in their seventh transmembrane domain.
• GPCRs activating PLD independently of ARF contain an AspProXXTyr motif.
• Mutating AspProXXTyr to AsnProXXTyr in GnRHR conferred ARF inhibitor sensitivity; reciprocal mutation in 5-HT2A receptor reduced sensitivity.

Conclusions:

• The AsnProXXTyr motif in GPCRs is critical for forming functional complexes with ARF and RhoA.
• This motif dictates the involvement of ARF and RhoA in GPCR-mediated phospholipase D activation.
• Structural variations in GPCRs determine their signaling pathways involving small G proteins.