<i>Gβγ</i> activates <i>PIP2</i> hydrolysis by recruiting and orienting <i>PLCβ</i> on the membrane surface

Abstract

Phospholipase C-βs (PLCβs) catalyze the hydrolysis of phosphatidylinositol 4, 5–bisphosphate <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mrow> <mml:mo>(</mml:mo> <mml:mi>P</mml:mi> <mml:mi>I</mml:mi> <mml:mi>P</mml:mi> <mml:mn>2</mml:mn> <mml:mo>)</mml:mo> </mml:mrow> </mml:math> into <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mrow> <mml:mi>inositoltriphosphate</mml:mi> </mml:mrow> </mml:math> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mrow> <mml:mo>(</mml:mo> <mml:mi mathvariant="italic">IP3</mml:mi> <mml:mo>)</mml:mo> </mml:mrow> </mml:math> and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mrow> <mml:mi mathvariant="normal">diacylglycerol</mml:mi> </mml:mrow> </mml:math> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mrow> <mml:mo>(</mml:mo> <mml:mi mathvariant="italic">DAG</mml:mi> <mml:mo>)</mml:mo> </mml:mrow> </mml:math> . <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mrow> <mml:mi>P</mml:mi> <mml:mi>I</mml:mi> <mml:mi>P</mml:mi> <mml:mn>2</mml:mn> </mml:mrow> </mml:math> regulates the activity of many membrane proteins, while IP3 and DAG lead to increased intracellular Ca 2+ levels and activate protein kinase C, respectively. PLCβs are regulated by G protein–coupled receptors through direct interaction with <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:msub> <mml:mrow> <mml:mi>G</mml:mi> <mml:mi>α</mml:mi> </mml:mrow> <mml:mi>q</mml:mi> </mml:msub> </mml:math> and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mi>G</mml:mi> <mml:mi>β</mml:mi> <mml:mi>γ</mml:mi> </mml:math> and are aqueous-soluble enzymes that must bind to the cell membrane to act on their lipid substrate. This study addresses the mechanism by which <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mi>G</mml:mi> <mml:mi>β</mml:mi> <mml:mi>γ</mml:mi> </mml:math> activates PLCβ 3. We show that PLCβ 3 functions as a slow Michaelis–Menten enzyme ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mrow> <mml:msub> <mml:mrow> <mml:msub> <mml:mi>k</mml:mi> <mml:mrow> <mml:mi mathvariant="italic">cat</mml:mi> </mml:mrow> </mml:msub> <mml:mo>∼</mml:mo> <mml:mn>2</mml:mn> <mml:mi> </mml:mi> <mml:msup> <mml:mrow> <mml:mi mathvariant="italic">s</mml:mi> </mml:mrow> <mml:mrow> <mml:mo>-</mml:mo> <mml:mn>1</mml:mn> </mml:mrow> </mml:msup> <mml:mo>,</mml:mo> <mml:mi> </mml:mi> <mml:mi>K</mml:mi> </mml:mrow> <mml:mi>M</mml:mi> </mml:msub> <mml:mo>∼</mml:mo> <mml:mn>0.43</mml:mn> <mml:mi> </mml:mi> <mml:mi>m</mml:mi> <mml:mi>o</mml:mi> <mml:mi>l</mml:mi> <mml:mo>%</mml:mo> </mml:mrow> </mml:math> ) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of PLCβ 3. Its partition coefficient is such that only a small quantity of PLCβ 3 exists in the membrane in the absence of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mi>G</mml:mi> <mml:mi>β</mml:mi> <mml:mi>γ</mml:mi> </mml:math> . When <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mi>G</mml:mi> <mml:mi>β</mml:mi> <mml:mi>γ</mml:mi> </mml:math> is present, equilibrium binding on the membrane surface increases PLCβ 3 in the membrane, increasing <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:msub> <mml:mi>V</mml:mi> <mml:mrow> <mml:mi mathvariant="italic">max</mml:mi> </mml:mrow> </mml:msub> </mml:math> in proportion. Atomic structures on membrane vesicle surfaces show that two <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mi>G</mml:mi> <mml:mi>β</mml:mi> <mml:mi>γ</mml:mi> </mml:math> anchor PLCβ 3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mi>G</mml:mi> <mml:mi>β</mml:mi> <mml:mi>γ</mml:mi> </mml:math> activates

Topics & Concepts